阅读说明：本技术 用于检测腺相关病毒(aav)载体转导的增强子和抑制剂和/或检测或定量抗aav结合抗体的体外测定 (In vitro assay for detecting enhancers and inhibitors of adeno-associated virus (AAV) vector transduction and/or for detecting or quantifying anti-AAV binding antibodies ) 是由 克劳迪亚·库兰达 泽维尔·安谷拉 费德里科·麦格兹 于 2019-11-15 设计创作，主要内容包括：本文公开了用于分析或检测来自受试者的生物样品中腺相关病毒(AAV)载体细胞转导的非抗体抑制剂和/或增强子的存在的方法。本文还公开了用于分析或检测抑制、降低或减少来自受试者的生物样品中的AAV载体细胞转导的AAV结合抗体的存在的方法。这些方法部分依赖于使用空衣壳AAV颗粒来吸收AAV结合抗体,以检测在分析AAV中和抗体(NAb)的生物样品中的AAV载体细胞转导的增强子或抑制剂(当存在时)。(Disclosed herein are methods for analyzing or detecting the presence of non-antibody inhibitors and/or enhancers of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject. Also disclosed herein are methods for analyzing or detecting the presence of an AAV binding antibody that inhibits, reduces or reduces transduction of AAV vector cells in a biological sample from a subject. These methods rely in part on the use of empty capsid AAV particles to absorb AAV binding antibodies to detect enhancers or inhibitors (when present) of AAV vector cell transduction in a biological sample analyzed for AAV neutralizing antibodies (nabs).)

1. a method for analyzing or detecting the presence of an enhancer of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein

(i) The vector comprises a reporter transgene comprising a reporter transgene,

(ii) the reporter transgene comprises a single strand or a self-complementing genome,

(iii) the reporter transgene is operably linked to one or more expression control elements; and

(iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing the infectious recombinant AAV particles of (a);

(f) providing a biological sample of a subject;

(g) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i);

(k) comparing the s.ev to the MAX, wherein if the s.ev is greater than the MAX, the biological sample from the subject comprises an enhancer transduced by AAV vector cells.

2. A method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein

(i) The vector comprises a reporter transgene comprising a reporter transgene,

(ii) the reporter transgene comprises a single strand or a self-complementing genome,

(iii) the reporter transgene is operably linked to one or more expression control elements; and

(iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing the infectious recombinant AAV particles of (a);

(f) providing a biological sample of a subject;

(g) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i);

(k) comparing the s.ev to the MAX, wherein if the s.ev is less than the MAX, the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector.

3. A method for analyzing or detecting the presence of an enhancer of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein

(i) The vector comprises a reporter transgene comprising a reporter transgene,

(ii) the reporter transgene comprises a single strand or a self-complementing genome,

(iii) the reporter transgene is operably linked to one or more expression control elements; and

(iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) providing a biological sample of a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(e) contacting the cell of (b) with the M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene;

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is expression of the reporter transgene in the absence of the sample from the subject and in the absence of addition of the empty capsid AAV particles, wherein if the expression of (f) is greater than the + control, then a biological sample from the subject comprises an AAV vector cell transduced enhancer.

4. A method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein

(i) The vector comprises a reporter transgene comprising a reporter transgene,

(ii) the reporter transgene comprises a single strand or a self-complementing genome,

(iii) the reporter transgene is operably linked to one or more expression control elements; and

(iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) providing a biological sample of a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(e) contacting the cell of (b) with the M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene;

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is the expression of the reporter transgene in the absence of the sample from the subject and in the absence of the addition of the empty capsid AAV particles, wherein if the expression of (f) is less than the + control, then a biological sample from the subject comprises an inhibitor of AAV vector cell transduction.

5. A method for analyzing, detecting, or quantifying an AAV binding antibody that inhibits AAV vector cell transduction in a biological sample from a subject, comprising:

(a) providing infectious recombinant AAV particles comprising a recombinant AAV vector, wherein

(i) The vector comprises a reporter transgene comprising a reporter transgene,

(ii) the reporter transgene comprises a single strand or a self-complementing genome,

(iii) the reporter transgene is operably linked to one or more expression control elements; and

(iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing the infectious recombinant AAV particles of (a);

(f) providing a diluted biological sample of a subject;

(g) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibodies present in the diluted biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i);

(k) providing the infectious recombinant AAV particles of (a);

(l) Providing a diluted biological sample from the same subject as the sample provided in (f);

(m) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(n) mixing the diluted biological sample of (i) with the infectious recombinant AAV particles of (k) to produce a mixture (M);

(o) contacting the cell of (M) with the M under conditions such that the infectious recombinant AAV particle of (k) can transduce the cell of (M) and express the reporter transgene in the cell of (M);

(p) measuring the expression of the reporter transgene and determining a value expressed as S reflecting the amount of reporter transgene expression of (o);

(q) performing steps (h) - (j) and (n) - (p) at least twice at different sample dilutions;

(r) wherein AAV binding antibodies that inhibit transduction of AAV vector cells are present in the diluted biological sample if S is less than MAX and s.ev is equal to or greater than MAX; and, optionally

(s) measuring the expression of a negative control of cells that can be infected with the infectious recombinant AAV particle of (a) but not with the infectious recombinant AAV particle of (a) to provide a background value expressed as MIN, wherein MIN can be subtracted from any of S, MAX and/or s.ev.

6. The method of claim 5, further comprising after step (r) or step (S), calculating a titer of AAV binding antibodies in the biological sample, the titer corresponding to a dilution that provides about 50% or more inhibition of reporter transgene expression, wherein the titer is a dilution that provides about 50% or more inhibition as determined by the formula S/MAX if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1:5, or wherein the titer is a dilution that provides about 50% or more inhibition as determined by the formula S/S.EV if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is less than about 1: 5.

7. The method of claim 5, further comprising the step (t) of calculating a titer of AAV binding antibodies in the biological sample that corresponds to providing about 50% or more inhibition of reporter transgene expression, wherein if the minimum dilution that provides about 50% or more inhibition of the expression of the reporter transgene is greater than or equal to about 1:5, the titer is the dilution that provides about 50% or more inhibition as determined by the formula 100- [ [ (S-MIN)/(MAX-MIN) ] × 100], or wherein if the minimum dilution that provides about 50% or more inhibition of the expression of the reporter transgene is less than about 1:5, the titer is the dilution that provides about 50% or more inhibition as determined by the formula 100- [ [ (S-MIN)/(s.ev-MIN) ] × 100 ].

8. The method of any one of claims 5-7, further comprising:

providing the infectious recombinant AAV particles of (a);

providing a cell that can be infected with the infectious recombinant AAV particle;

providing an empty capsid AAV particle;

contacting the cell with the provided empty capsid AAV particles;

contacting the cell that has been contacted with the empty capsid AAV particle with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell;

measuring the expression of the reporter gene and determining a value expressed as max.ev reflecting the expression amount of the reporter gene.

9. The method of any of claims 5-8, further comprising calculating a signal-to-noise ratio, expressed as S/N, where S/N is MAX/MIN.

10. The method according to any one of claims 5-9, further comprising calculating a coefficient of variation percentage (% CV), wherein% CV (standard deviation/mean) x 100%.

11. The method according to any of claims 5-10, further comprising calculating EV interference, wherein EV interference is MAX/MAX.

12. The method of any one of claims 5-12, further comprising:

(a) measuring the expression of the reporter gene under control conditions comprising one or more dilutions of AAV binding antibody that bind to AAV vector, and determining a value expressed as HQC for the measurement of expression at said dilution that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN; and/or

(b) Measuring expression of the reporter transgene under control conditions comprising the dilution of step (a) that provides a preselected amount of expression of the reporter transgene relative to MAX or MAX-MIN in the presence of empty capsid AAV particles, and determining a value expressed as hqc.

13. The method of claim 12, wherein the preselected amount of reporter transgene expression of step (a) is equal to or less than about 30% of MAX or MAX-MIN.

14. The method of claim 12 or 13, further comprising calculating EV efficacy, wherein EV efficacy is HQC.

15. The method of any one of claims 5-13, wherein steps (h) - (j) and (n) - (p) are performed at least 3, 4, 5, or 6 times at 3, 4, 5, or 6 different dilutions of the biological sample.

16. The method of any one of claims 5-14, wherein the biological sample is diluted to between about 1:1 to about 1:1000 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

17. The method of any one of claims 5-16, wherein steps (h) - (j) and (n) - (p) are performed at a dilution of about 1:1, about 1:2.5, about 1:5, about 1:10, about 1:100, and/or about 1:1000 of the biological sample.

18. The method of any one of claims 1-16, wherein the method is completed within about 48 hours of step (c) or (d).

19. The method according to any one of claims 1-18, wherein the cells that can be infected with the infectious recombinant AAV particles are inoculated from a frozen cell aliquot.

20. The method according to any one of claims 1-19, wherein the cells of any one of steps (c), (e), (i), or (o) that can be infected with the infectious recombinant AAV particle are contacted within about 2 hours after they are thawed from freezing.

21. The method of any one of claims 1-20, wherein the cells of any one of steps (c), (e), (i), or (o) that are capable of being infected with the infectious recombinant AAV particle are at least about 80% confluent.

22. A carrier or plate having individually disposed thereon:

(a) a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, and the infectious recombinant AAV particle comprising the reporter transgene;

(b) a diluted biological sample from the subject, cells capable of infection by the infectious recombinant AAV particles, and empty capsid AAV particles; and

(c) a diluted biological sample from the same subject that provided the sample of (b), a cell that can be infected with an infectious recombinant AAV particle comprising a reporter transgene, and the infectious recombinant AAV particle comprising the reporter transgene.

23. The carrier or plate of claim 22, wherein the carrier or plate comprises plastic or glass.

24. The carrier or plate of claim 22 or 23, wherein the carrier or plate is a multi-well plate or tray.

25. The carrier or plate of any one of claims 22-24, wherein each of (a), (b), and (c) is arranged within a separate tube or a separate single well of a porous carrier or plate.

26. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the cells capable of being infected with the infectious recombinant AAV particles comprise mammalian cells.

27. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the cell is capable of infection by an AAV particle comprising VP1, VP2, or VP3 sequences having 90% or more identity to VP1, VP2, or VP3 sequences of a hybrid or chimera of any one of the foregoing AAV serotypes, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or a VP3 sequence of a hybrid or chimera of any one of the foregoing AAV serotypes.

28. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the cell is capable of infection by an AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or a hybrid or chimera of any of the foregoing AAV serotypes.

29. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the cells capable of being infected with the infectious recombinant AAV particles comprise 2V6.11, HEK-293, CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, a549, HT1080, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, or HEL cells, optionally wherein any of the la cells express an adenovirus E4 gene.

30. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene encodes a secreted or a secretable protein.

31. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent, or electrochemical signal.

32. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene comprises a luciferase gene.

33. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene comprises a renilla luciferase, a firefly luciferase, or a gaussian luciferase gene.

34. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene comprises a β -galactosidase gene, a β -glucuronidase gene, or a chloramphenicol transferase gene.

35. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene encodes Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), or alkaline phosphatase.

36. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the expression control element comprises a promoter and/or enhancer nucleic acid sequence operable in a mammalian cell.

37. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the expression control element comprises CAG (SEQ ID NO:3), Cytomegalovirus (CMV) immediate early promoter/enhancer, Rous Sarcoma Virus (RSV) promoter/enhancer, SV40 promoter, dihydrofolate reductase (DHFR) promoter, chicken β -actin (CBA) promoter, phosphoglycerate kinase (PGK) promoter, or elongation factor-1 α (EF1- α) promoter.

38. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the flanking elements comprise one or more AAV Inverted Terminal Repeats (ITRs).

39. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the reporter transgene is located between one or more 5 'and/or 3' AAVITR.

40. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the flanking elements comprise mutant, modified or variant AAV ITRs that are not processed by AAV Rep proteins.

41. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the flanking elements are included in the infectious recombinant AAV particles are mutant, modified, or variant AAV ITRs that allow or promote formation of a double stranded inverted repeat structure from a complementary reporter transgene genome.

42. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the mutant, modified or variant AAV ITRs have a deleted D sequence, and/or a mutant, modified or variant Terminal Resolution Site (TRS) sequence.

43. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the recombinant vector comprises a first Inverted Terminal Repeat (ITR) of AAV; a promoter operable in a mammalian cell; the reporter transgene; a polyadenylation signal; and optionally a second ITR of AAV.

44. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the recombinant vector comprises a restriction site and a post-transcriptional regulatory element downstream of the restriction site that enable insertion of the reporter transgene downstream of a promoter operable in mammalian cells, wherein the promoter, the restriction site, and the post-transcriptional regulatory element are located downstream of a 5'AAV ITR and optionally upstream of a 3' AAV ITR.

45. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the infectious recombinant AAV particles comprise an AAV serotype that infects primates.

46. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the infectious recombinant AAV particles comprise an AAV serotype that infects humans or rhesus monkeys.

47. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the infectious recombinant AAV particle comprises a VP1, VP2, or VP3 sequence that is 90% or more identical to a VP1, VP2, or VP sequence of a hybrid or chimera of an AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or any of the above AAV serotypes.

48. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the infectious recombinant AAV particle comprises AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74, Rh10, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or a hybrid or chimera of any of the above AAV serotypes.

49. The method of any one of claims 1-21 or the carrier or plate of any one of claims 22-25, wherein the biological sample comprises a primate sample.

50. The method of any one of claims 1-21 or the carrier or plate of any one of claims 22-25, wherein the biological sample comprises serum.

51. The method of any one of claims 1-21 or the carrier or plate of any one of claims 22-25, wherein the biological sample comprises plasma.

52. The method according to any one of claims 1-21 or the carrier or plate according to any one of claims 22-25, wherein the biological sample comprises human serum or human plasma.

53. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject is a mammal.

54. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject is a human, said human optionally having a genetic disease treatable by gene therapy.

55. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject has a disorder due to insufficient expression or activity of a protein.

56. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject has a condition that is caused by abnormal, or undesirable expression or activity of a protein.

57. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject has a genetic disease.

58. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject is a candidate for gene replacement or supplemental therapy.

59. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject is a candidate for gene knockdown or knock-out therapy.

60. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the subject has a lung disease (e.g., cystic fibrosis), a bleeding disorder (e.g., hemophilia a or hemophilia B with or without inhibitors), thalassemia, a hematologic disorder (e.g., anemia), alzheimer's disease, parkinson's disease, huntington's disease, Amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage disease (e.g., aspartyl diabetes, barton's disease, cystinosis, late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), fabry disease, gaucher's disease I, II and type III, glycogen storage disease type II, pompe disease, GM 2-type I gangliosidosis (tay-saxosis), gangliosidosis type 2-II (sandofur disease), Mucolipidosis type I (salivary gland diseases type I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudoherlerian disease) and mucolipidosis type IV, mucopolysaccharidosis (herlerian disease and variants, hunter, sanfilippo type A, B, C, D, morqui types a and B, malachite-gardney disease), niemann-pick types a/B, C1 and C2, and sinderler types I and II), Hereditary Angioedema (HAE), copper or iron accumulation disorders (e.g., wilson or menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), retinal degenerative diseases (e.g., RPE65 disease), Choroideremia, Stargardt disease and other eye diseases), solid organ (e.g., brain, liver, kidney, heart) diseases or infectious virus (e.g., hepatitis b and c, HIV, etc.), bacterial or fungal diseases.

61. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, further comprising inputting information about the presence or titer of AAV binding antibodies in the biological sample into a report associated with the subject.

62. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, further comprising inputting information about the presence or titer of AAV binding antibodies in the biological sample into a database, thereby generating a database entry, the database entry being associated with the subject.

63. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the AAV binding antibody analyzed or detected binds to AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV3B, AAV-2i8, Rh74 or Rh10, or a hybrid or chimera of any of the foregoing AAV serotypes.

64. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the biological sample is diluted prior to contacting with the infectious recombinant AAV particles of (a), (e) or (k).

65. The method of any one of claims 1-21 or the carrier or plate of any one of claims 22-25, wherein the biological sample is analyzed for multiple dilutions.

66. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the biological sample is analyzed for a plurality of different dilution ratios.

67. The method of any one of claims 1-21 or the vehicle or plate of any one of claims 22-25, wherein the cells capable of being infected with the infectious recombinant AAV particles are not lysed prior to measuring expression of the reporter transgene.

68. The method of any one of claims 1-21 or the carrier or plate of any one of claims 22-25, wherein the biological sample is heat inactivated.

69. The method of claim 1, wherein one or more of steps (c), (d), (h), (i), (j), or (k) is performed with an automated system.

70. The method of claim 69, wherein the automated system comprises a contact assembly, a measurement assembly, a mixing assembly, an incubation assembly, a processor, and non-transitory electronic storage configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly, the method further comprising:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device; and

(k) comparing, with the processor, the s.ev to the MAX, wherein if the s.ev is greater than the MAX, the processor determines that the biological sample from the subject comprises an enhancer transduced by AAV vector cells, and optionally outputting, with the processor, an indication that the biological sample from the subject comprises an enhancer for display.

71. The method of claim 2, wherein one or more of steps (c), (d), (h), (i), (j), or (k) is performed with an automated system.

72. The method of claim 71, wherein the automated system comprises a contact assembly, a measurement assembly, a mixing assembly, an incubation assembly, a processor, and non-transitory electronic storage configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly, the method further comprising:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device; and

(k) comparing, with the processor, the s.ev to the MAX, wherein if the s.ev is less than the MAX, the processor determines that the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputs, with the processor, an indication that the biological sample from the subject comprises an inhibitor for display.

73. The method of claim 3, wherein one or more of steps (d), (e), (f) or (g) is performed with an automated system.

74. The method of claim 73, wherein the automated system comprises a contact assembly, a measurement assembly, a mixing assembly, an incubation assembly, a processor, and non-transitory electronic storage configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly, the method further comprising:

(d) mixing the biological sample of (c) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(e) contacting the cell of (b) and the infectious recombinant AAV particle of (a) with the contact component under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene with the measurement component; and

(g) comparing the expression of (f) to the positive (+) control with the processor, wherein if the expression of (f) is greater than the + control, the processor determines that the biological sample from the subject comprises an enhancer of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputs with the processor an indication that the biological sample from the subject comprises the enhancer for display.

75. The method of claim 4, wherein one or more of steps (d), (e), (f), or (g) is performed with an automated system.

76. The method of claim 75, wherein the automated system comprises a contact assembly, a measurement assembly, a mixing assembly, an incubation assembly, a processor, and non-transitory electronic storage configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly, the method further comprising:

(d) mixing the biological sample of (c) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(e) contacting the cell of (b) and the infectious recombinant AAV particle of (a) with the contact component under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene with the measurement component; and

(g) comparing the expression of (f) to the positive (+) control with the processor, wherein if the expression of (f) is less than the + control, the processor determines that the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputs with the processor an indication that the biological sample from the subject comprises the inhibitor for display.

77. The method of claim 5, wherein one or more of steps (c), (d), (h), (i), (j), (n), (o), (p), or(s) is performed with an automated system.

78. The method of claim 77, wherein the automated system comprises a contact assembly, a measurement assembly, a mixing assembly, an incubation assembly, a processor, and non-transitory electronic storage configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly, the method further comprising:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the diluted biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device;

(n) mixing the diluted biological sample of (i) with the infectious recombinant AAV particles of (e) with the mixing component to produce the mixture M;

(o) contacting the cell of (M) with the M with the contacting means under conditions such that the infectious recombinant AAV particle of (k) transduces the cell of (M) and expresses the reporter transgene in the cell of (M);

(p) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted S that reflects the amount of reporter transgene expression of (o), and storing with the processor the value denoted S in the non-transitory electronic storage device; and

(s) optionally measuring with the measuring component the expression of a negative control of the cell that can be infected with the infectious recombinant AAV particle (a) but not infected with the infectious recombinant AAV particle (a) to provide a background value expressed as MIN, wherein MIN can be subtracted by the processor from any of S, MAX and/or s.ev.

79. The method of claim 78, further comprising steps(s) or (t),

calculating a titer of the AAV binding antibody, the titer corresponding to a minimum dilution of the biological sample that provides about 50% or more inhibition of reporter transgene expression, with the processor configured such that the titer is determined by the formula S/MAX if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1:5, or by the formula S/s.ev if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is less than about 1: 5; and

optionally, outputting, with the processor, for display, an indication of the titer.

80. The method of claim 78, further comprising step (t),

calculating a titer of the AAV binding antibody with the processor, the titer corresponding to a minimum dilution of the biological sample that provides about 50% or more inhibition of reporter transgene expression, wherein the processor is configured such that the titer is determined by the formula 100- [ [ (S-MIN)/(MAX-MIN) ]. times.100 ] if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1:5, or by the formula 100- [ [ (S-MIN)/(S.EV-MIN) ]. times.100 ] if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is less than about 1: 5; and

optionally, outputting, with the processor, for display, an indication of the titer.

81. The method of any one of claims 78-80, further comprising:

providing an infectious recombinant AAV particle (a);

providing a cell that is capable of being infected with the infectious recombinant AAV particle;

providing an empty capsid AAV particle;

contacting the cell with the provided empty capsid AAV particles with the contacting component;

contacting the cell that has been contacted with the empty capsid AAV particle with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell;

measuring expression of the reporter transgene with the measurement component, determining the value expressed as max.ev that reflects the amount of reporter transgene expression with the processor, and storing the value expressed as max.ev in the non-transitory electronic storage device with the processor.

82. The method of any of claims 78-81, further comprising calculating with the processor a signal-to-noise ratio represented as S/N, wherein the S/N is equal to MAX/MIN, storing with the processor S/N in the non-transitory electronic storage device, and optionally outputting with the processor an indication of S/N for display.

83. The method of any of claims 78-82, further comprising calculating, with the processor, a coefficient of variation percentage (% CV), wherein% CV (standard deviation/average) x 100%, storing, with the processor,% CV in the non-transitory electronic storage device, and optionally outputting, with the processor, an indication of% CV for display.

84. The method of any of claims 78-83, further comprising calculating, with the processor, EV interference, where EV interference is MAX/MAX.

85. The method of any one of claims 78-84, further comprising calculating HQC, using the processor, based on expression measurements that provide the dilution at which transgene expression is reported relative to a preselected amount of MAX or MAX-MIN; and/or calculating HQC EV based on expression of the reporter transgene under control conditions comprising the dilution of step (a) that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles; and/or calculating HQC EV/HQC, storing HQC and/or HQC EV/HQC with the processor in the non-transitory electronic storage device, and optionally outputting an indication/HQC of HQC and/or HQC EV with the processor for display.

Background

Adeno-associated virus (AAV) vector gene transfer has demonstrated clinical efficacy in human clinical trials for the treatment of Leber congenital amaurosis and for hemorrhagic diseases a and B. As a result of exposure to wild-type AAV, a varying percentage of humans will exhibit capsid-bound antibodies, which can inhibit or prevent AAV vector cell transduction. Such antibodies that bind to AAV are a major obstacle to AAV-based gene therapy vectors, rendering some patients unable to obtain potentially life-saving therapies. Thus, subjects positive for neutralizing antibodies (nabs) of AAV are usually excluded from gene therapy trials and are not optimal candidates for gene therapy.

anti-AAV antibodies can be measured using a binding assay, wherein IgG binding to the virus is detected, or using a cell transduction inhibition assay, wherein the cell transduction efficiency of the reporter vector is measured in vitro. Although antibody binding assays are easy to set up, they cannot determine which binding antibodies will affect AAV vector transduction. In contrast, cell-based NAb assays do measure the degree of inhibition of vector transduction mediated by anti-AAV circulating factors, but are time consuming and challenging with respect to sensitivity and accuracy. Furthermore, lack of standardization in the procedure of the Nab assay is an obstacle to interpreting gene therapy trial results.

Disclosure of Invention

As disclosed herein, there are other factors, different from AAV binding antibodies, that can inhibit, reduce, or reduce AAV vector cell transduction. As also disclosed herein, there are factors that can enhance transduction of AAV vector cells. These enhancing and inhibiting factors may be present in certain subjects suitable for treatment with or participating in clinical trials of AAV-based gene therapy. Typically, a subject is assessed for the presence of AAV-binding antibodies to determine the suitability/eligibility for gene therapy treatment. The presence or absence of AAV-binding antibodies in a subject can also be assessed after receiving gene therapy treatment to monitor the development of antibodies or subsequent re-administration of gene therapy treatment. However, if a subject in need of measuring AAV-binding antibodies has an enhancer or inhibitor of AAV vector cell transduction, a typical cell-based antibody assay will provide inaccurate quantitative results regarding AAV-binding antibody titers.

For example, in the presence of factors that enhance transduction of AAV vector cells in a subject with AAV-binding antibodies, the amount of AAV-binding antibodies will be underestimated, and if low enough will result in false negatives of AAV-binding antibodies in the subject. Such false negative subjects are actually positive for AAV-binding antibodies.

In the presence of an agent that inhibits AAV vector cell transduction in a subject, even in the absence of detectable AAV binding antibodies, if sufficient inhibitor is present to inhibit or reduce AAV vector cell transduction, the test will result in false positives for AAV binding antibodies in the subject. Such false positive subjects are actually negative for AAV binding antibodies or have relatively low AAV antibody titers.

Thus, the invention provides, inter alia, methods of assaying for the presence of such an enhancer or inhibitor in a sample and methods of detecting the presence of such an enhancer or inhibitor in a sample. Such as a biological sample from a subject.

In certain embodiments, a method for analyzing or detecting the presence of an enhancer transduced by adeno-associated virus (AAV) vector cells in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector comprising a reporter transgene operably linked to one or more expression regulatory elements; b. an empty capsid AAV particle, c. cells permissive for AAV infection and d. a biological sample from a subject;

(b) the first measurement was performed by: a. contacting the cell with the infectious recombinant AAV particle under conditions such that the cell is transduced by the infectious recombinant AAV particle and the reporter transgene is expressed by the cell; measuring the expression of the reporter transgene and determining a value expressed as MAX;

(c) the second measurement was performed by: a. mixing the biological sample with empty capsid AAV particles to produce a mixture (M), and incubating the M under conditions such that the empty capsid AAV particles can bind any AAV binding antibodies present in the biological sample; b. contacting the cell with the M and the infectious recombinant AAV particle under conditions such that the cell is transduced by the infectious recombinant AAV particle and a reporter gene is expressed by the cell; measuring the expression of the reporter transgene and determining a value expressed as s.ev; and

(d) comparing the s.ev to the MAX, wherein if the s.ev is greater than the MAX, the biological sample from the subject comprises an enhancer transduced by AAV vector cells.

In certain embodiments, a method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector comprising a reporter transgene operably linked to one or more expression regulatory elements; b. an empty capsid AAV particle, c. cells permissive for AAV infection and d. a biological sample from a subject;

(b) the first measurement was performed by: a. contacting the cell with the infectious recombinant AAV particle under conditions such that the cell is transduced by the infectious recombinant AAV particle and the reporter transgene is expressed by the cell; measuring the expression of the reporter transgene and determining a value expressed as MAX;

(c) the second measurement was performed by: a. mixing the biological sample with empty capsid AAV particles to produce a mixture (M), and incubating the M under conditions such that the empty capsid AAV particles can bind any AAV binding antibodies present in the biological sample; b. contacting the cell with the M and the infectious recombinant AAV particle under conditions such that the cell is transduced by the infectious recombinant AAV particle and a reporter gene is expressed by the cell; measuring the expression of the reporter transgene and determining a value expressed as s.ev; and

(d) comparing the s.ev to the MAX, wherein if the s.ev is less than the MAX, the biological sample from the subject comprises an inhibitor of AAV vector cell transduction.

In certain embodiments, a method for analyzing or detecting the presence of an enhancer transduced by adeno-associated virus (AAV) vector cells in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the expression of the transgene is reported by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing an infectious recombinant AAV particle (a);

(f) providing a biological sample from a subject;

(g) providing a cell that can be infected with an infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of M and (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene of the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i); and

(k) comparing the s.ev to MAX, wherein if the s.ev is greater than MAX, the biological sample from the subject comprises an enhancer transduced by AAV vector cells.

In certain embodiments, a method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the expression of the transgene is reported by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing an infectious recombinant AAV particle (a);

(f) providing a biological sample from a subject;

(g) providing a cell that can be infected with an infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of M and (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene of the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i); and

(k) comparing the s.ev to MAX, wherein if the s.ev is less than MAX, the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector.

In certain embodiments, a method for analyzing or detecting the presence of an enhancer transduced by adeno-associated virus (AAV) vector cells in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) providing a biological sample from a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(e) contacting the cell of (b) with M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) is capable of transducing the cell of (b) and expresses the reporter transgene of the cell of (b);

(f) measuring expression of the reporter transgene;

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is expression of the reporter transgene, no sample from the subject and no empty capsid AAV particles are added, wherein if (f) is greater than the + control, the biological sample from the subject contains an enhancer transduced by AAV vector cells.

In certain embodiments, a method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) providing a biological sample from a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(e) contacting the cell of (b) with M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) is capable of transducing the cell of (b) and expresses the reporter transgene of the cell of (b);

(f) measuring expression of the reporter transgene;

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is expression of the reporter transgene, no sample from the subject and no empty capsid AAV particles are added, wherein if (f) is less than the + control, the biological sample from the subject contains an inhibitor of AAV vector cell transduction.

The invention also provides, inter alia, methods for analyzing, detecting, or quantifying an AAV binding antibody that inhibits, reduces, or reduces transduction of AAV vector cells in a sample, e.g., a biological sample from a subject.

In certain embodiments, a method for analyzing, detecting, or quantifying an AAV binding antibody that inhibits AAV vector cell transduction in a biological sample from a subject comprises:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the expression of the transgene is reported by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing an infectious recombinant AAV particle (a);

(f) providing a diluted biological sample from a subject;

(g) providing a cell that can be infected with an infectious recombinant AAV particle;

(h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of M and (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene of the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i);

(k) providing an infectious recombinant AAV particle (a);

(l) Providing a diluted biological sample from the same subject as the sample provided in (f)

(m) providing a cell capable of being infected with an infectious recombinant AAV particle;

(n) mixing the diluted biological sample of (l) with the infectious recombinant AAV particles of (k) to produce a mixture (M);

(o) contacting the cell of (M) with M under conditions such that the infectious recombinant AAV particle of (k) is capable of transducing the cell of (M) and expressing the reporter transgene in the cell of (M);

(p) measuring the expression of the reporter transgene and determining a value expressed as S reflecting the amount of reporter transgene expression of (o);

(q) performing steps (h) - (j) and (n) - (p) at least twice at different sample dilutions;

(r) wherein AAV binding antibodies that inhibit transduction of AAV vector cells are present in the diluted sample if S is less than MAX and s.ev is equal to or greater than MAX; and, optionally

(s) measuring the expression of a negative control of cells capable of being infected with the infectious recombinant AAV particle (a) but not infected with the infectious recombinant AAV particle (a) to provide a background value expressed as MIN, wherein MIN can be subtracted from any of S, MAX and/or s.ev.

In certain embodiments, the method steps may be performed in any suitable order unless otherwise indicated herein.

In certain embodiments, the method further comprises, after step (r) or after step (S), calculating a titer of AAV binding antibodies in the biological sample, the titer corresponding to providing about 50% or more inhibition of reporter gene expression, wherein the titer is the inhibition of the dilution providing about 50% or more inhibition as determined by the formula S/MAX if the minimum dilution providing about 50% or more inhibition of reporter gene expression is greater than or equal to about 1:5, or wherein the titer is the dilution providing about 50% or more inhibition as determined by the formula S/s.ev if the minimum dilution providing about 50% or more inhibition of reporter gene expression is less than about 1: 5.

In certain embodiments, the method further comprises the step (t) of calculating a titer of AAV binding antibody in the biological sample, the titer corresponding to providing about 50% or more inhibition of reporter gene expression, wherein the titer is an inhibition of a dilution providing about 50% or more inhibition as determined by the formula 100- [ [ (S-MIN)/(MAX-MIN) ] × 100] if the minimum dilution providing about 50% or more inhibition of reporter gene expression is greater than or equal to about 1:5, or wherein the titer is a dilution providing about 50% or more inhibition as determined by the formula 100- [ [ (S-MIN)/(s.ev-MIN) ] × 100] if the minimum dilution providing about 50% or more inhibition of reporter gene expression is less than about 1: 5.

In certain embodiments, the method further comprises: providing an infectious recombinant AAV particle (a); providing a cell that can be infected with an infectious recombinant AAV particle; providing an empty capsid AAV particle; contacting a cell with the provided empty capsid AAV particles; contacting a cell that has been contacted with an empty capsid AAV particle with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and a reporter gene for expression by the cell; and measuring the expression of the reporter gene and determining a value expressed as max.ev that reflects the amount of reporter gene expression.

In certain embodiments, the method further comprises calculating a signal-to-noise ratio, expressed as S/N, where S/N equals MAX/MIN.

In certain embodiments, a method further comprises calculating a percentage coefficient of variation (% CV), wherein% CV is (standard deviation/mean) x 100%.

In certain embodiments, a method further comprises calculating an EV interference, wherein the EV interference is MAX/MAX.

In certain embodiments, a method further comprises: (a) measuring the expression of the reporter gene under control conditions comprising one or more dilutions of AAV binding antibody that bind to AAV vector, and determining a value expressed as HQC for the measurement of expression at said dilution that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN; and/or (b) measuring expression of the reporter transgene under control conditions comprising the dilution of step (a) that provides a preselected amount of expression of the reporter transgene relative to MAX or MAX-MIN in the presence of empty capsid AAV particles, and determining a value expressed as hqc.

The preselected amount of reporter transgene expression of step (a) may be equal to or less than about 75% of MAX or MAX-MIN, such as, but not limited to, equal to or less than about 60% of MAX or MAX-MIN, equal to or less than about 50% of MAX or MAX-MIN, equal to or less than about 40% of MAX or MAX-MIN, equal to or less than about 30% of MAX or MAX-MIN. In certain embodiments, the preselected amount of reporter transgene expression of step (a) is equal to or less than about 25% of MAX or MAX-MIN. In certain embodiments, the preselected amount of reporter transgene expression of step (a) is equal to or less than about 20% of MAX or MAX-MIN.

In certain embodiments, a method further comprises calculating EV efficacy, wherein EV efficacy is HQC.

In certain embodiments, steps (h) - (j) and (n) - (p) are performed at least 3, 4, 5, or 6 times at 3, 4, 5, or 6 different dilutions of the biological sample.

In certain embodiments, the sample is diluted between about 1:1 and about 1:5000 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

In certain embodiments, the sample is diluted between about 1:1 to about 1:1000 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

In certain embodiments, the sample is diluted between about 1:1 to about 1:500 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

In certain embodiments, the sample is diluted between about 1:1 and about 1:100 prior to contacting or incubating with the infectious recombinant AAV particles of (a), (e) or (k).

In certain embodiments, steps (h) - (j) and (n) - (p) are performed at a dilution of about 1:1, about 1:2.5, about 1:5, about 1:10, about 1:100, and/or about 1:1000 of the biological sample.

In certain embodiments, the method is completed at the beginning of step (c) or (d) or within about 48 hours after completion of step (c) or (d).

In certain embodiments, cells that can be infected with the infectious recombinant AAV particle of any of steps (c), (e), (i), or (o) are contacted within about 2 hours after thawing the cells that can be infected with the infectious recombinant AAV particle from freezing.

In certain embodiments, cells that can be infected with the infectious recombinant AAV particle of any of steps (c), (e), (i), or (o) are contacted within about 1 hour after the infected cells are thawed from frozen after the cells that can be infected with the infectious recombinant AAV particle.

In certain embodiments, cells that are capable of being infected with the infectious recombinant AAV particle of any one of steps (c), (e), (i), or (o) are at least about 60% confluent.

In certain embodiments, cells that are capable of being infected with the infectious recombinant AAV particle of any one of steps (c), (e), (i), or (o) are at least about 70% confluent.

In certain embodiments, cells that are capable of being infected with the infectious recombinant AAV particle of any one of steps (c), (e), (i), or (o) are at least about 80% confluent.

In certain embodiments, cells that are capable of being infected with the infectious recombinant AAV particle of any one of steps (c), (e), (i), or (o) are at least about 90% confluent.

The invention also provides, inter alia, carriers and plates having one or more components for use in the method of the invention. The vehicles and plates can include, for example, without limitation, cells that can be infected with infectious recombinant AAV particles, a biological sample from a subject, such as a diluted biological sample from a subject, and/or empty capsid AAV particles.

In certain embodiments, the carrier or plate has separately disposed thereon:

(a) a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, an infectious recombinant AAV particle comprising a reporter transgene;

(b) a diluted biological sample from the subject, cells capable of infection by infectious recombinant AAV particles and empty capsid AAV particles; and/or

(c) A diluted biological sample from the same subject as the sample providing (b), a cell capable of being infected with an infectious recombinant AAV particle comprising a reporter transgene, and an infectious recombinant AAV particle comprising a reporter transgene.

In certain embodiments, each component on the carrier or plate may be in a separate well. In certain embodiments, each component on the carrier or plate may be in an amount disclosed herein.

In certain embodiments, the carrier or plate has each of the (a), (b), and (c) components disposed within individual tubes or individual cells of the porous carrier or plate.

In certain embodiments, the carrier or plate comprises plastic or glass.

In certain embodiments, the carrier or plate is a multi-well plate or tray.

In certain embodiments, a method for analyzing or detecting the presence of an enhancer transduced by adeno-associated virus (AAV) vector cells in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the expression of the transgene is reported by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing an infectious recombinant AAV particle (a);

(f) providing a biological sample from a subject;

(g) providing a cell that can be infected with an infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of M and (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene of the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i); and

(k) comparing the s.ev to MAX, wherein if the s.ev is greater than MAX, the biological sample from the subject comprises an enhancer transduced by AAV vector cells.

In certain embodiments, one or more of steps (c), (d), (h), (i), (j), or (k) may be performed with an automated system.

In certain embodiments, the automated system comprises a contact component, a measurement component, a mixing component, an incubation component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly. The method further comprises:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device; and

(k) comparing, with the processor, the s.ev to the MAX, wherein if the s.ev is greater than the MAX, the processor determines that the biological sample from the subject comprises an enhancer transduced by AAV vector cells, and optionally outputting, with the processor, an indication that the biological sample from the subject comprises an enhancer for display.

In certain embodiments, a method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the expression of the transgene is reported by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing an infectious recombinant AAV particle (a);

(f) providing a biological sample from a subject;

(g) providing a cell that can be infected with an infectious recombinant AAV particle;

(h) mixing the biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles are capable of binding to any AAV binding antibodies present in the biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of M and (e) under conditions such that the infectious recombinant AAV particles of (e) are capable of transducing the cell of (g) and expressing the reporter transgene of the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i); and

(k) comparing the s.ev to MAX, wherein if the s.ev is less than MAX, the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector.

In certain embodiments, one or more of steps (c), (d), (h), (i), (j), or (k) may be performed with an automated system.

In certain embodiments, the automated system comprises a contact component, a measurement component, a mixing component, an incubation component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly. The method further comprises:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device; and

(k) comparing, with the processor, the s.ev to the MAX, wherein if the s.ev is less than the MAX, the processor determines that the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression and secretion of a protein encoded by the vector, and optionally outputs, with the processor, an indication that the biological sample from the subject comprises an inhibitor for display.

In certain embodiments, a method for analyzing or detecting the presence of an enhancer transduced by adeno-associated virus (AAV) vector cells in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) providing a biological sample from a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(e) contacting the cell of (b) with the M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene; and

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is expression of the reporter transgene in the absence of the sample from the subject and in the absence of addition of the empty capsid AAV particles, wherein if the expression of (f) is greater than the + control, then a biological sample from the subject comprises an AAV vector cell transduced enhancer.

In certain embodiments, one or more of steps (d), (e), (f), or (g) may be performed with an automated system.

In certain embodiments, the automated system comprises a contact component, a measurement component, a mixing component, an incubation component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly. The method further comprises

(d) Mixing the biological sample of (c) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(e) contacting the cell of (b) and the infectious recombinant AAV particle of (a) with the contact component under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene with the measurement component; and

(g) comparing the expression of (f) to the positive (+) control with the processor, wherein if the expression of (f) is greater than the + control, the processor determines that the biological sample from the subject comprises an enhancer of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputs with the processor an indication that the biological sample from the subject comprises the enhancer for display.

In certain embodiments, a method for analyzing or detecting the presence of an inhibitor of adeno-associated virus (AAV) vector cell transduction in a biological sample from a subject, comprising:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression control elements, and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that can be infected with an infectious recombinant AAV particle;

(c) providing a biological sample from a subject;

(d) mixing the biological sample of (c) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibody present in the biological sample;

(e) contacting the cell of (b) with the M and the infectious recombinant AAV particle of (a) under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene; and

(g) comparing the expression of (f) to a positive (+) control, wherein the + control is the expression of the reporter transgene in the absence of the sample from the subject and in the absence of the addition of the empty capsid AAV particles, wherein if the expression of (f) is less than the + control, then a biological sample from the subject comprises an inhibitor of AAV vector cell transduction.

In certain embodiments, one or more of steps (d), (e), (f), or (g) may be performed with an automated system.

In certain embodiments, the automated system comprises a contact component, a measurement component, a mixing component, an incubation component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly. The method further comprises:

(d) mixing the biological sample of (c) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(e) contacting the cell of (b) and the infectious recombinant AAV particle of (a) with the contact component under conditions such that the infectious recombinant AAV particle of (a) can transduce the cell of (b) and express the reporter transgene in the cell of (b);

(f) measuring expression of the reporter transgene with the measurement component; and

(g) comparing the expression of (f) to the positive (+) control with the processor, wherein if the expression of (f) is less than the + control, the processor determines that the biological sample from the subject comprises an inhibitor of AAV vector cell transduction, expression or secretion of a protein encoded by the vector, and optionally outputs with the processor an indication that the biological sample from the subject comprises the inhibitor for display.

As described herein, in certain embodiments, a method for analyzing, detecting, or quantifying an AAV binding antibody that inhibits AAV vector cell transduction in a biological sample from a subject, comprises:

(a) providing an infectious recombinant AAV particle comprising a recombinant AAV vector, wherein (i) the vector comprises a reporter transgene, (ii) the reporter transgene comprises a single stranded or self-complementary genome, (iii) the reporter transgene is operably linked to one or more expression regulatory elements; and (iv) the reporter transgene is flanked by one or more flanking elements;

(b) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene and determining a value expressed as MAX that reflects the amount of reporter transgene expression of (c);

(e) providing the infectious recombinant AAV particles of (a);

(f) providing a diluted biological sample of a subject;

(g) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(h) mixing the diluted biological sample of (f) with empty capsid AAV particles to produce a mixture (M) and incubating the M under conditions such that the empty capsid AAV particles can bind to any AAV binding antibodies present in the diluted biological sample;

(i) contacting the cell of (g) with the infectious recombinant AAV particles of (e) under conditions such that the infectious recombinant AAV particles of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene and determining a value expressed as s.ev that reflects the amount of reporter transgene expression of (i);

(k) providing the infectious recombinant AAV particles of (a);

(l) Providing a diluted biological sample from the same subject as the sample provided in (f);

(m) providing a cell that is capable of being infected with the infectious recombinant AAV particle;

(n) mixing the diluted biological sample of (i) with the infectious recombinant AAV particles of (k) to produce a mixture (M);

(o) contacting the cell of (M) with the M under conditions such that the infectious recombinant AAV particle of (k) can transduce the cell of (M) and express the reporter transgene in the cell of (M);

(p) measuring the expression of the reporter transgene and determining a value expressed as S reflecting the amount of reporter transgene expression of (o);

(q) performing steps (h) - (j) and (n) - (p) at least twice at different sample dilutions;

(r) wherein AAV binding antibodies that inhibit transduction of AAV vector cells are present in the diluted sample if S is less than MAX and s.ev is equal to or greater than MAX; and, optionally

(s) measuring the expression of a negative control of cells that can be infected with the infectious recombinant AAV particle (a) but not infected with the infectious recombinant AAV particle (a) to provide a background value expressed as MIN, wherein MIN can be subtracted from any of S, MAX and/or s.ev.

In certain embodiments, one or more of steps (c), (d), (h), (i), (j), (n), (o), (p), or(s) may be performed with an automated system.

In certain embodiments, the automated system comprises a contact component, a measurement component, a mixing component, an incubation component, a processor, and a non-transitory electronic storage device. The non-transitory electronic storage device is configured to cause the processor to control the contact assembly, the measurement assembly, the mixing assembly, and the incubation assembly. The method further comprises:

(c) contacting the cell of (b) with the infectious recombinant AAV particle of (a) with the contact component under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell of (b);

(d) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted MAX that reflects the amount of reporter transgene expression of (c), and storing with the processor the value denoted MAX in the non-transitory electronic storage device;

(h) mixing the diluted biological sample of (f) with the empty capsid AAV particles with the mixing component to produce the mixture M, and incubating the M with the incubation component under conditions such that the empty capsid AAV particles are capable of binding any AAV binding antibodies present in the biological sample that inhibit, reduce, or reduce AAV vector cell transduction;

(i) contacting the cell of (g) and the infectious recombinant AAV particle of (e) with the contact component under conditions such that the infectious recombinant AAV particle of (e) can transduce the cell of (g) and express the reporter transgene in the cell of (g);

(j) (ii) measuring the expression of the reporter transgene with the measurement component, determining with the processor the value expressed as s.ev that reflects the amount of reporter transgene expression of (i), and storing with the processor the value expressed as s.ev in the non-transitory electronic storage device;

(n) mixing the diluted biological sample of (i) with the infectious recombinant AAV particles of (e) with the mixing component to produce the mixture M;

(o) contacting the cell of (M) with the M with the contacting means under conditions such that the infectious recombinant AAV particle of (k) transduces the cell of (M) and expresses the reporter transgene in the cell of (M);

(p) measuring the expression of the reporter transgene with the measurement component, determining with the processor a value denoted S that reflects the amount of reporter transgene expression of (o), and storing with the processor the value denoted S in the non-transitory electronic storage device; and

(s) optionally measuring with the measuring component the expression of a negative control of the cell that can be infected with the infectious recombinant AAV particle (a) but not infected with the infectious recombinant AAV particle (a) to provide a background value expressed as MIN, wherein MIN can be subtracted by the processor from any of S, MAX and/or s.ev.

In certain embodiments, the method may further comprise the step of (S) or (t), calculating with the processor a titer of the AAV binding antibody, the titer corresponding to a minimum dilution of the biological sample that provides about 50% or more inhibition of reporter transgene expression, wherein the processor is configured such that the titer is determined by the formula S/MAX if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1:5, or by the formula S/s.ev if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is less than about 1: 5; and optionally, outputting, with the processor, for display, an indication of the titer.

In certain embodiments, the method can include the step (t) of calculating with the processor a titer of the AAV binding antibody, the titer corresponding to a minimum dilution of the biological sample that provides about 50% or more inhibition of reporter transgene expression, wherein the processor is configured such that the titer is determined by the formula 100- [ [ (S-MIN)/(MAX-MIN) ] × 100] if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is greater than or equal to about 1:5, or by the formula 100- [ [ (S-MIN)/(s.ev-MIN) ] × 100] if the minimum dilution that provides about 50% or more inhibition of reporter transgene expression is less than about 1: 5; and optionally, outputting, with the processor, for display, an indication of the titer.

In certain embodiments, the method further comprises providing an infectious recombinant AAV particle (a); providing a cell that is capable of being infected with the infectious recombinant AAV particle; providing an empty capsid AAV particle; contacting the cell with the provided empty capsid AAV particles with the contacting component; contacting the cell that has been contacted with the empty capsid AAV particle with the infectious recombinant AAV particle of (a) under conditions such that the cell of (b) is transduced by the infectious recombinant AAV particle of (a) and the reporter transgene is expressed by the cell; measuring expression of the reporter transgene with the measurement component, determining the value expressed as max.ev that reflects the amount of reporter transgene expression with the processor, and storing the value expressed as max.ev in the non-transitory electronic storage device with the processor.

In certain embodiments, the method further comprises calculating, with the processor, a signal-to-noise ratio represented as S/N, wherein the S/N is equal to MAX/MIN, storing, with the processor, the S/N in the non-transitory electronic storage device, and optionally outputting, with the processor, for display, an indication of the S/N.

In certain embodiments, the method further comprises calculating, with the processor, a percent coefficient of variation (% CV), wherein% CV is (standard deviation/mean) x 100%, storing, with the processor, the% CV in the non-transitory electronic storage device, and optionally outputting, with the processor, an indication of the% CV for display.

In certain embodiments, the method further comprises calculating, with the processor, an EV interference, wherein the EV interference is MAX/MAX.

In certain embodiments, the method further comprises calculating HQC, using the processor, based on the expression measurement value providing the dilution of reporter transgene expression relative to a preselected amount of MAX or MAX-MIN; and/or calculating HQC EV based on expression of the reporter transgene under control conditions comprising the dilution of step (a) that provides a preselected amount of reporter transgene expression relative to MAX or MAX-MIN in the presence of empty capsid AAV particles; and/or calculating HQC EV/HQC, storing HQC and/or HQC EV/HQC with the processor in the non-transitory electronic storage device, and optionally outputting an indication/HQC of HQC and/or HQC EV with the processor for display.

Drawings

FIG. 1A shows that 27% of healthy donor (HD; normal without any known disease) samples had enhancer (E), which resulted in a wrong estimate of AAV NAb titers in these samples. Also shown are 49% of HD with >1:40 AAV NAb titers, 14% of HD with 1:1 to 1:20 AAV NAb titers, 10% of HD AAV NAb negative.

Figure 1B shows that in the presence of enhancer, calculation of AAV NAb titers was inaccurate. "BAb" is an AAV binding antibody.

Figure 2 shows the detection of AAV nabs in serum using empty capsid AAV particles (EV).

Fig. 3A-3E show the results of a2 day assay using preincubation with empty capsid AAV particles (EV). X-axis titers were 1:1, 1:2.5, 1:5, 1:10, 1:100, and 1: 1000. A) NAb⁺(1:10)；B)NAb^-(<1:1)；C)NAb^-(<1:1), an enhancer; D) NAb^-(<1:1), inhibitor, false positive; E) NAb⁺(1:1), enhancer, false negative.

Figure 4 shows a decision tree for AAV NAb titer calculation.

FIGS. 5A and 5B show representative formulas for calculating A)% inhibition (MAX) and B)% inhibition (SEV). MIN background.

Figures 6A-6E show examples of AAV NAb titer calculations. A) Assigning a decision tree for AAV NAb titer based on test sample dilution; B) -a sample; C) low NAb titer; D) an enhancer in the sample; E) high NAb titer.

Fig. 7 shows an exemplary pickup board layout.

FIGS. 8A-8C show the detection criteria (1). A) Signal to noise ratio (S/N); B) EV efficacy; C) and (4) EV interference.

FIGS. 9A-9C show the detection criteria (2).

FIG. 10A shows an exemplary FACT dilution scheme (wells 3B-3D).

FIG. 10B shows an exemplary sample dilution scheme (wells 4B-4G).

FIG. 11 shows an exemplary transfer of diluted sample to a neutralization plate

Fig. 12 shows an exemplary addition of FBS to the neutralization plate (hole: MIN 2B; S4B-4G; s.ev 5B-5G; FACT 3B-3D; FACT 3E, 3F; MAX 2G; and max.ev 3G).

Fig. 13A shows an exemplary addition of empty capsid AAV particles to a neutralization plate (well: MIN 2B; S4B-4G; s.ev 5B-5G; FACT 3B-3D; fact.ev 3E, 3F; MAX 2G; and max.ev 3G).

Fig. 13B shows an exemplary addition of DMEM medium to the neutralization plate (well: MIN 2B; S4B-4G; s.ev 5B-5G; FACT 3B-3D; fact.ev 3E, 3F; MAX 2G; and max.ev 3G).

Figure 14 shows an exemplary addition of AAV reporter vector to the neutralization plate (well: MIN 2B; S4B-4G; s.ev 5B-5G; FACT 3B-3D; fact.ev 3E, 3F; MAX 2G; and max.ev 3G).

Fig. 15 shows controls and samples transferred from the neutralization plate to the transfer plate (wells: MIN 2B-2D; S2E-2G; s.ev 9B-9G, 10B-10G and 11B-11G; FACT 3B-3D, 4B-4D and 5B-5D; FACT 3E, 3F, 4E, 4F, 5E and 5F; MAX 2E-2G; and MAX 3G, 4G and 5G).

FIG. 16 shows a schematic representation of the CAG promoter sequence (SEQ ID NO:3) consisting of the Cytomegalovirus (CMV) early enhancer element ("C"), the first exon and the first intron of the chicken β -actin gene ("A"), and the splice acceptor of the rabbit β -globin gene ("G").

Detailed Description

Disclosed herein are methods of detecting enhancers and inhibitors of AAV vector transduction, as well as methods for detecting and/or quantifying AAV binding antibodies (also referred to as neutralizing antibodies (nabs)) that inhibit, reduce, or reduce transduction of AAV vector cells. The methods according to the invention rely in part on transduction of an AAV permissive cell line with a reporter vector (an AAV particle carrying a reporter transgene). The methods according to the invention also rely in part on the use of empty capsid AAV particles to absorb most or all of the AAV binding antibodies in a sample from a subject, revealing the presence of enhancers or inhibitors (if any) of AAV vector cell transduction in the sample analyzed for AAV NAb. The methods according to the invention are particularly useful for more accurately determining AAV NAb titers in samples from subjects, where the presence of an enhancer or inhibitor can result in false negatives and false positives, respectively.

In certain embodiments, certain steps in the assay method according to the invention are optimized. In certain embodiments, the time to completion of the assay is reduced. In certain embodiments, assay variability is reduced and/or matrix interference is reduced. In particular, for example, the assay method according to the invention can be performed in about 2 days instead of 3 days, the intra-assay variation assessed by the coefficient of variation (% CV) of three repeated measurements is reduced from 30.5% to 8.7%, and for quality control samples, the inter-assay precision assessment of the assay shows a% CV of 12.5%, compared to conventional NAb assays (Meliani et al, 2015, Human Gene Therapy Methods, 26:45-53, doi: 10.1089/hgtb.2015.037).

The method according to the invention provides a simplified, reliable and more accurate assay that can be used to detect or quantify AAV binding antibodies (AAV neutralizing antibodies (NAbs)) that inhibit, reduce or reduce transduction of AAV vector cells in various circumstances. For example, in certain embodiments, the assay can be used to analyze, detect, or quantify AAV nabs to support selection of a subject for gene therapy treatment or to exclude a subject from gene therapy treatment. In certain embodiments, the assay can be used to analyze, detect, or quantify AAV nabs, to select subjects or to exclude subjects from gene therapy trials. In certain embodiments, the assay can be used to analyze, detect, or quantify AAV nabs to monitor the development of anti-AAV antibodies in a subject after receiving gene therapy treatment. In certain embodiments, the assay can be used to analyze, detect, or quantify AAV nabs to monitor AAV nabs in a subject that may need treatment or has received treatment to reduce the amount of AAV nabs.

In certain embodiments, a method is provided for analyzing, detecting, or quantifying an AAV binding antibody that inhibits, reduces, or reduces transduction of AAV vector cells in a biological sample from a subject.

In certain embodiments, a method is provided for analyzing, detecting, or quantifying an AAV neutralizing antibody that inhibits, reduces, or reduces transduction of AAV vector cells in a biological sample from a subject.

Adeno-associated virus (AAV) vectors are viral vectors that infect, inter alia, primates, such as humans.

As used herein and without limitation, the term "recombinant" as a modifier of a vector (such as a recombinant aav (raav) vector) and of a sequence (such as a recombinant polynucleotide and polypeptide) means that the composition has been manipulated (i.e., engineered) in a manner that does not normally occur in nature. Specific examples of recombinant AAV vectors would be: wherein a nucleic acid not normally found in a wild-type AAV genome (heterologous polynucleotide) is inserted into the viral genome. Examples thereof would be: wherein a nucleic acid (e.g., a gene) encoding a reporter transgene is cloned into a vector. Although the term "recombinant" is not always used herein with reference to AAV vectors and sequences such as transgenes, recombinant forms, including AAV vectors, polynucleotides, and the like, are also expressly included, despite any such omissions.

For example, a "rAAV vector" is derived from a wild-type AAV genome by using molecular methods to remove all or a portion of the wild-type AAV genome and replace with a non-native (heterologous) nucleic acid encoding a reporter transgene, such as a reporter protein. Typically, one or both Inverted Terminal Repeat (ITR) sequences of the AAV genome are retained for rAAV vectors. rAAV differs from AAV genomes because all or a portion of the AAV genome has been replaced with a non-native sequence, relative to the AAV genomic nucleic acid, such as with a reporter transgene encoding a reporter protein. Thus, the incorporation of non-native (heterologous) sequences defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector.

The recombinant AAV vector sequences may be packaged, referred to herein as "particles," for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo. Where the recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle may also be referred to as a "rAAV," rAAV particle, "and/or a" rAAV virion. Such rAAV, rAAV particles, and rAAV virions include proteins that encapsidate or package the vector genome. In the case of AAV, particular embodiments include capsid proteins.

As used herein, "infectious recombinant AAV particles" refers to packaged recombinant AAV vector sequences that can infect or transduce a cell ex vivo, in vitro, or in vivo. As used herein, "cells capable of being infected" refers to cells infected or transduced with infectious recombinant AAV particles. In certain embodiments, the methods of the invention employ cells that can be infected with infectious rAAV particles in vitro.

The "vector genome," which may be abbreviated as "vg," refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a rAAV particle. In the case of recombinant plasmids used to construct or make recombinant AAV vectors, the AAV vector genome does not include portions of the "plasmid" that do not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genomic portion of the recombinant plasmid is referred to as the "plasmid backbone" that is essential for cloning and amplification of the plasmid, a process required for propagation and production of the recombinant AAV vector, but is not itself packaged or encapsidated into rAAV particles. Thus, "vector genome" refers to a nucleic acid packaged or encapsidated by a rAAV

Empty capsid AAV particles (EV) or Empty Vectors (EV) refer to AAV particles that lack a vector genome. Empty capsid AAV particles can be used to absorb (bind) AAV binding antibodies to analyze or detect the presence of enhancers of AAV vector cell transduction or inhibitors of AAV vector cell transduction. Empty capsid AAV particles can also be used to quantify AAV NAb titers at relatively low titers (e.g., less than about 1: 5).

As used herein, the term "serotype" with respect to an AAV vector refers to a capsid that is serologically distinct from other AAV serotypes. Serodiscrimination is determined based on the lack of cross-reactivity between the antibody and one AAV compared to another AAV. The cross-reactivity differences are typically attributed to differences in capsid protein sequences/antigenic determinants (e.g., differences in VP1, VP2, and/or VP3 sequences attributed to AAV serotypes). Due to homology of capsid protein sequences or similar or identical conformational epitopes, antibodies directed against one AAV capsid serotype may cross-react with one or more other AAV capsid serotypes.

Under the traditional definition, serotype means that the virus of interest has been tested for neutralizing activity against sera of all existing and characteristic serotypes, and no antibodies found to neutralize the virus of interest. There may or may not be a serological difference from any currently existing serotype due to the discovery of more naturally occurring viral isolates and/or the generation of capsid mutants. Thus, in the absence of serological differences in a new virus (e.g., AAV), the new virus (e.g., AAV) will be a subgroup or variant of the corresponding serotype. In many cases, mutant viruses with capsid sequence modifications have not been serologically tested for neutralizing activity to determine whether they belong to another serotype according to the traditional serotype definition. Thus, for convenience and to avoid repetition, the term "serotype" broadly refers to both a serologically distinct virus (e.g., AAV) and a serologically distinct virus (e.g., AAV) that may be within a subgroup or variant of a given serotype.

rAAV vectors and empty capsid AAV particles include any strain or serotype. For example, and without limitation, an AAV vector genome or particle (capsid, such as VP1, VP2, and/or VP3) can be based on any AAV serotype, such as, for example, AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, AA3B, or AAV-2i 8. These vectors and empty capsid AAV particles may be based on the same virus strain or serotype (or subgroup or variant), or different from each other. For example, and without limitation, a rAAV vector genome or particle (capsid) based on one serotype genome may be identical to one or more of the capsid proteins of the packaging vector. In addition, the rAAV vector genome can be based on an AAV serotype genome that is different from one or more of the capsid proteins packaging the vector genome, in which case at least one of the three capsid proteins can be a different AAV serotype, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or variants thereof. More specifically, the rAAV2 vector genome may comprise AAV2 ITRs in addition to capsids from different serotypes, such as, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), or variants thereof. Thus, rAAV vectors include gene/protein sequences that have the same characteristics as the gene/protein sequences of a particular serotype, as well as "mixed" serotypes, also referred to as "pseudotypes".

In certain embodiments, the AAV vector comprising the reporter gene has a capsid serotype that is the same as a capsid serotype of the empty capsid AAV particle. However, as long as the empty capsid AAV particles are capable of absorbing (binding) antibodies bound by the AAV vector comprising the reporter gene, for example due to cross-reactivity, the capsid serotype of the empty capsid AAV particles need not be the same serotype as the capsid serotype of the AAV vector comprising the reporter gene.

In certain embodiments, the rAAV vector or empty capsid AAV particle comprises or consists of a capsid sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2) capsid proteins (VP1, VP2, and/or VP3 sequences). In certain embodiments, the rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, or-rh 10 ITRs.

In certain embodiments, rAAV vectors or empty capsid AAV particles include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV3B, and AAV-2i8 variants (e.g., capsid variants, such as amino acid insertions, rAAV additions, substitutions, and deletions in the context of a rAAV vector, and ITR nucleotide insertions, additions, substitutions, and deletions), e.g., as set forth in WO 2013/158879 (international application PCT/US 2013/037170), WO 2015/013313 (international application PCT/US 2014/047670), and US 2013/0059732 (U.S. application No.13/594,773), thereof.

rAAV and empty capsid AAV particles (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV3B, AAV-2i8, SPK1(SEQ ID NO:1), SPK2(SEQ ID NO:2), and variants, hybrids, and chimerics) can be constructed using recombinant techniques known to those of skill in the art to include one or more heterologous polynucleotide sequences (transgenes) flanked at the 5 'and/or 3' ends with one or more functional AAV ITR sequences. rAAV vectors typically retain at least one functional flanking ITR sequence as needed for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include sequences required in cis for replication and packaging (e.g., functional ITR sequences)

In certain embodiments, the AAV vector is used to transduce a target cell with a reporter transgene, which is subsequently transcribed and optionally translated, thereby providing a detectable signal to detect or quantify transgene expression. The amount of signal is directly proportional to the efficiency of cell transduction and subsequent expression. An antibody that binds to a carrier protein that packages or encapsulates the reporter transgene or inhibitor will inhibit, reduce or diminish the amount of transduction of the carrier cell, subsequent expression of the reporter gene, and a detectable signal.

In the assays described herein, for the analysis, detection and quantification of antibodies, selection of the particular capsid protein serotype packaging or encasing the reporter transgene can be used to identify the serotype to which the NAb binds. For example, if it is desired to detect AAV-2 antibodies, the reporter transgene can be encapsulated by AAV-2 capsid proteins. If it is desired to detect AAV-8 antibodies, the reporter transgene can be encapsulated by AAV-8 capsid proteins. If it is desired to detect AAV-9 antibodies, the reporter transgene can be encapsulated by AAV-9 capsid proteins. If present, the antibody binds to the capsid protein encapsulating the reporter transgene, thereby inhibiting, reducing or diminishing vector cell transduction and subsequent expression of the reporter transgene. The greater the amount or titer of antibody bound to the envelope or capsid protein, the less vector transduction and consequent reporter transgene expression and signaling by the cell. Thus, the methods herein for analyzing, detecting, and quantifying antibodies that bind to a carrier protein (e.g., a viral (e.g., AAV) capsid protein) can also be used to identify the presence or absence of antibodies that bind to any particular AAV capsid serotype.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced or unspliced mRNA, rRNA tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microrna (mirna), small or short interfering (si) RNA, trans-spliced RNA, or antisense RNA).

Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The nucleic acid may be single-stranded, double-stranded or triple-stranded, linear or circular, and may be of any length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to convention that sequences are provided in the 5 'to 3' direction.

A "heterologous" transgene or nucleic acid refers to a polynucleotide inserted into a vector (AAV) for the purpose of vector-mediated transfer/delivery of the polynucleotide into a cell. The heterologous transgene is distinct from the vector (AAV) nucleic acid, i.e., non-native with respect to the viral (AAV) nucleic acid sequence. Once transferred/delivered into the cell, the heterologous transgene contained within the virion can be expressed (e.g., transcribed and translated, if appropriate). Although the term "heterologous" is not always used herein to refer to a transgene or nucleic acid, reference to a transgene or nucleic acid is meant to include a heterologous transgene or nucleic acid, even in the absence of the modifier "heterologous," although omitted.

As used herein, a "reporter" transgene is a polynucleotide that provides a detectable signal. The detectable signal can be provided by the reporter transgene itself, the transcript of the transgene, or by a protein encoded by the reporter transgene.

Transgenes in all mammalian and non-mammalian forms are expressly included, including but not limited to the examples disclosed herein that report transgenes and encoded proteins, whether known or unknown. Thus, the methods according to the invention include reporter transgenes and proteins from microorganisms and other organisms that are detectable in the cell following transduction or transfer as described herein.

In certain embodiments, the reporter transgene encodes a secreted or a secretable protein. In certain embodiments, the reporter transgene encodes a protein that provides an enzymatic, colorimetric, fluorescent, luminescent, chemiluminescent, or electrochemical signal.

For example, but not limited to, a reporter transgene includes a luciferase gene encoding a luciferase protein, such as, but not limited to, a Renilla luciferase, a firefly luciferase, or a Gaussian luciferase gene.

For example, but not limited to, the reporter transgene may encode beta-galactosidase, beta-glucuronidase, chloramphenicol transferase, Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), and alkaline phosphatase.

"polypeptide", "protein" and "peptide" encoded by a "transgene" include full-length native sequences, as well as naturally occurring proteins, as well as functional subsequences, modifications or sequence variants, so long as the subsequences, modifications or variants retain some degree of function of the native full-length protein. In the present invention, these polypeptides, proteins and peptides encoded by the transgene may, but need not, be identical to the wild-type protein.

In certain embodiments, the reporter transgene (which transgene provides a detectable signal) may comprise a single strand or a self-complementary genome. Self-complementing transgenes become double-stranded or double-stranded dimers when packaged into viral particles (e.g., AAV vectors) or when transduced in a viral vector cell and the virus uncoats in the transduced cell.

The term "complementary" or "complementary" when used in reference to a nucleic acid, such as a transgene, refers to a plurality of chemical bases such that one single-stranded sequence does or is capable of "specifically hybridizing" or binding (annealing) to another single-stranded sequence to form a double-stranded or double-stranded molecule through base pairing. The ability of two single-stranded sequences to specifically hybridize or bind (anneal) to each other and form a double-stranded (or duplex) molecule relies on the functional group of a base on one strand (e.g., sense) that will form a hydrogen bond with another base on the opposite nucleic acid strand (e.g., antisense). Complementary bases capable of binding to each other are usually A and T, and C and G in DNA, and C and G, and U and A in RNA. Thus, an example of a self-complementary sequence may be ATCGXXXCGAT, X representing a non-complementary base, such that the structure of a duplex or duplex with unhybridised X bases behaves as:

the terms "complementary" and "complementary" when used in reference to a polynucleotide or nucleic acid, such as a transgene, are therefore intended to describe the physical state of formation of a double stranded or duplex polynucleotide or nucleic acid molecule, or simply the sequence relationship between two polynucleotides or nucleic acid molecules, such that each single stranded molecule can form a double strand with the other. Thus, "complementary" and "complementary" refer to the base relationship of each polynucleotide or nucleic acid molecule strand, and not to the fact that the two strands must be present in a duplex (or duplex) configuration or physical state with respect to each other.

Typically, for viral vectors that package single-stranded nucleic acids, such as AAV, Inverted Terminal Repeat (ITR) sequences are involved in replication and form hairpin loops, which aid in self priming, thereby allowing initiation and synthesis of the second DNA strand. After synthesis of the second DNA strand, the AAV ITRs have a so-called Terminal Resolution Site (TRS), such that the hairpin loop is cleaved into two single strands, each having 5 'and 3' terminal repeats, for viral packaging.

The use of a deleted, mutated, modified or non-functional TRS in at least one ITR results in the formation of a double-stranded duplex that is not cleaved at the TRS. For self-complementary reporter transgenic double stranded duplex structures, there is typically an ITR with a deleted, mutated or variant TRS between the two complementary strands. Non-cleavable or unresolvable TRS enable the formation of self-complementary reporter transgene duplex structures because the double stranded form is not cleaved. Or unresolvable itres with TRSs with a confirmation, mutation or variation, or resolvable itres may be suitable for viral packaging. Resolvable AAV ITRs need not be wild-type ITR sequences, as long as the ITRs mediate the desired function (e.g., packaging, self-priming, replication, etc.).

The ITR and TRS sequences of the various AAV serotypes that may be deleted, mutated, modified or altered include any of the AAV serotypes described herein or known to those of skill in the art. For example, but not limited to, the ITR and TRS sequences of the various AAV serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -rh74, -rh 10. Another example is a modified or mutated AAV ITR that is not processed by AAV Rep proteins. Another example is a mutated, modified or variant AAV ITR with a deleted D sequence and/or a mutated, modified or variant Terminal Resolution Site (TRS) sequence. For AAV2, representative mutant TRS sequences are: "CGGTTG".

For self-complementary reporter transgene sequences located outside, e.g., one or more ITRs, expression control sequences, downstream sequences, etc., such sequences in the vector sequence outside the reporter transgene may, but need not, be self-complementary. Thus, self-complementation may be used in a particular context, e.g., to refer to a transgene, e.g., a reporter transgene, such that only the transgene (e.g., the reporter transgene) is self-complementary, while other non-transgene sequences may, but need not, be self-complementary.

In order to be self-complementary, not all bases in a single strand must be complementary to every base of the opposite complementary strand. Only a sufficient number of complementary nucleotides or nucleobases are required to enable two polynucleotides or nucleic acid molecules to specifically hybridize or bind (anneal) to each other. Thus, short sequence segments or regions of non-complementary bases will exist between self-complementary polynucleotides or nucleic acid molecules. For example, but not limited to, 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, or 100 and 150 or more contiguous or non-contiguous non-complementary bases may be present, but sufficient complementary bases will be present over the length of the two sequences to enable the two polynucleotides or nucleic acid molecules to specifically hybridize or bind (anneal) to each other and form a double-stranded (or duplex) sequence. Thus, the sequences of the two single stranded regions may be less than 100% complementary to each other, but still capable of forming a double stranded molecule. In certain embodiments, the two single stranded sequences have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more mutual complementarity.

Such segments or regions of non-complementary bases between self-complementary polynucleotides or nucleic acid molecules may be internal sequences such that when complementary portions of two single-stranded molecules form a double strand or duplex, the non-complementary bases form a circular or convex structure, the overall structure resembling a hairpin. Such segments or regions of non-complementary bases between self-complementary polynucleotides or nucleic acid molecules may also flank the complementary region, in which case one or both of the 5' or 3 flanking regions may not form a double stranded duplex.

In cells with a transgene, the transgene has been introduced/transferred by a vector, such as a viral vector (e.g., AAV). This process is referred to as "transduction" or "transfection" of the cell. The terms "transduction" and "transfection" refer to the introduction of a molecule (e.g., a transgene) into a cell.

Cells into which a transgene has been introduced are referred to as "transduced or transfected" cells. Thus, a "transduced" or "transfected" cell refers to a change in the cell following incorporation of an exogenous molecule, such as a polynucleotide (e.g., an AAV vector comprising a transgene), into the cell. Thus, for example, a "transduced" or "transfected" cell is a cell or progeny thereof into which an exogenous molecule has been introduced. The cells can be propagated and the introduced transgene and/or expressed protein transcribed.

The cell that can be the target of transduction by the vector with the transgene (e.g., a viral vector) can be any cell that is susceptible to infection or can be infected by the vector. Such cells include mammalian cells. These cells may have low, medium or high susceptibility to infection. Thus, target cells include cells of any tissue or organ type, of any origin (e.g., mesodermal, ectodermal, or endodermal). Examples of cells that may be infected and that may be used in the method according to the invention include, for example, but are not limited to, liver (e.g. hepatocytes, antral endothelial cells), pancreas (e.g. pancreatic beta islet cells), lung, central or peripheral nervous system, such as brain (e.g. nerve, glial or ependymal cells) or spine, kidney (HEK-293 cells), eye (e.g. retina, cellular components), spleen, skin, thymus, testis, lung, diaphragm, heart (cardiac), muscle or psoas muscle, or intestinal tract (e.g. endocrine glands), adipose tissue (white, brown or beige), muscle (e.g. fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nerve cells, or hematopoietic (e.g. blood or lymph) cells.

In certain embodiments, cells that can be targets transduced with a vector (e.g., a viral vector) carrying a transgene can be seeded from frozen cell aliquots or from cell banks. In certain embodiments, cells that can be targets transduced by a vector (e.g., a viral vector) with a transgene can be inoculated from cultured cells.

Examples of cell lines that can be targeted for transduction by vectors with transgenes (e.g., viral vectors) include, for example, but are not limited to, 2V6.11, HEK-293, CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, and HeLa cells.

In certain embodiments, cells that can be targeted for transduction with a vector bearing a transgene (e.g., a viral vector) can transiently or stably express the E4 gene from an adenovirus. E4 gene expression ensures efficient cell transduction of AAV vectors.

AAV vectors and vector sequences may include one or more "expression control elements" or "expression regulatory elements. Typically, an expression control or regulatory element is a nucleic acid sequence that affects the expression of an operably linked polynucleotide (e.g., a transgene). Control elements present in the vector, including expression control and regulatory elements as described herein, e.g., promoters and enhancers, are included to facilitate appropriate transgene transcription and, if appropriate, translation (e.g., promoters, enhancers, splicing signals for introns, maintaining the correct reading frame for the polynucleotide to allow in-frame translation of mRNA and stop codons, etc.). These elements generally function in cis, but may also function in trans.

Expression control can be achieved at the level of transcription, translation, splicing, message stability, and the like. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end of the transcribed polynucleotide (i.e., "upstream"). Expression control elements may also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., in an intron). The expression control element can be located at a distance from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, 5000 to 10,000 or more nucleotides from the polynucleotide), or even a substantial distance. However, due to the limitation of the length of the polynucleotide, such expression control elements are typically in the range of 1 to 1000 nucleotides from the polynucleotide for AAV vectors.

Functionally, expression of an operably linked transgene is controlled at least in part by the element, such that the element regulates transcription of the polynucleotide and, where appropriate, translation of the transcript. One specific example of an expression control element is a promoter, which is typically located at the 5' end of a transcribed sequence. Another example of an expression control element is an enhancer, which may be located at the 5 'or 3' end of a transcribed sequence, or within a transcribed sequence.

As used herein, "promoter" may refer to a DNA sequence that is generally adjacent to a transgene. A promoter generally increases the amount of expression from a transgene compared to the amount of expression in the absence of the promoter.

As used herein, "enhancer" may refer to a sequence adjacent to a transgene. Enhancer elements are typically located upstream of a promoter element, but also function within a DNA sequence (e.g., a transgene), and may be located downstream or within a DNA sequence (e.g., a transgene). Thus, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a transgene. Enhancer elements generally increase expression of the transgene above that provided by the promoter element.

Examples of expression regulatory or expression control elements that can be used in the methods according to the present invention include, for example, but are not limited to, CAG (SEQ ID NO:3), Cytomegalovirus (CMV) immediate early promoter/enhancer, Rous Sarcoma Virus (RSV) promoter/enhancer, SV40 promoter, dihydrofolate reductase (DHFR) promoter, chicken β -actin (CBA) promoter, phosphoglycerate kinase (PGK) promoter, and elongation factor-1 α (EF1- α) promoter.

Antibodies (which may be referred to as "neutralizing" antibodies) that bind to recombinant viral vectors (e.g., rAAV vectors) useful for gene therapy can reduce, inhibit, or reduce cellular transduction of the viral vector. As a result, although not being bound by theory, cell transduction is reduced, inhibited or reduced, thereby reducing the introduction and subsequent expression and, where appropriate, subsequent translation into protein or peptide of the virally packaged heterologous polynucleotide into the cell.

An immune response, e.g., humoral immunity, can be generated against the wild-type virus in a subject exposed to the wild-type virus. Such exposure may result in binding of pre-existing antibodies in the subject to a wild-type virus-based viral vector, even prior to treatment with a gene therapy method using the viral vector. Alternatively, antibodies can be produced in a subject following treatment with a recombinant viral vector or following exposure to a wild-type virus.

Biological samples are typically obtained from or produced by biological organisms. Examples of biological samples from a subject that can be analyzed using the methods according to the present invention include, for example, but are not limited to, whole blood, serum, plasma, and the like, and combinations thereof. Other biological samples from a subject that may be used in a method according to the invention include, for example, but are not limited to, cerebrospinal fluid or simply spinal fluid. The biological sample may be free of cells, or may include cells (e.g., red blood cells, platelets, and/or lymphocytes).

Suitable subjects from which biological samples for analysis can be obtained by using the method according to the invention include mammals, such as primates (e.g. humans), as well as non-human mammals. The term "subject" refers to an animal, typically a mammal, such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), a farm animal (dogs and cats), a farm animal (poultry (such as chicken and ducks), horses, cattle, goats, sheep, pigs), and a laboratory animal (mouse, rat, rabbit, guinea pig). Suitable human subjects include fetal, neonatal, infant, juvenile and adult subjects. Subjects also include animal disease models, such as mice and other animal models, such as non-human primates.

Suitable subjects (e.g., humans) whose biological samples can be analyzed using the methods according to the present invention also include, for example, but are not limited to, those having loss-of-function and gain-of-function genetic diseases, disorders, and deficiencies. Thus, a subject (e.g., a human) includes a subject that is a candidate for or is receiving gene replacement or supplementation therapy (e.g., protein/enzyme replacement therapy), as well as a subject (e.g., a human) that is a candidate for or is receiving gene knockdown or knockout therapy (e.g., a human).

As used herein, the term "loss of function" with respect to a genetic defect refers to any mutation in a gene in which the protein encoded by the gene (i.e., the mutein) exhibits a partial or complete loss of function normally associated with the wild-type protein. This includes any disease, disorder or defect caused or contributed to by insufficient expression or activity of the protein.

As used herein, the term "gain of function" with respect to a genetic defect refers to any mutation in a gene in which the protein encoded by the gene (i.e., the mutant protein) gains a function not normally associated with the protein (i.e., the wild-type protein) that results in or contributes to a disease or disorder. The gain-of-function mutation may be a deletion, addition or substitution of one or more nucleotides in a gene, which may result in an alteration of the function of the encoded protein. Gain-of-function mutations can alter the function of a mutant protein or cause interactions with other proteins. Gain-of-function mutations can also result in the reduction or elimination of a normal wild-type protein, for example, by interaction of an altered mutant protein with a normal wild-type protein. Gain-of-function mutations can result in a condition or disease caused or caused by abnormal, or undesirable expression or activity of a protein.

Suitable human subjects whose biological samples can be analyzed using the methods according to the invention include, for example, but are not limited to, subjects having a genetic disease or genetic disorder treatable by gene therapy. Gene therapy treatments or therapies include vector (e.g., viral vectors, such as AAV vectors) mediated delivery of nucleic acids for the treatment of diseases or disorders.

Suitable human subjects whose biological samples can be analyzed using the method according to the invention also include, for example and without limitation, subjects suffering from: pulmonary diseases (e.g., cystic fibrosis), bleeding disorders (e.g., hemophilia a or hemophilia B with or without inhibitors), thalassemia, blood disorders (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartyl diabetes, barton disease, advanced infantile ceroid lipofuscinosis type 2 (CLN2), cystinosis, Fabry disease, Gaucher disease I, II and Gaucher disease type III (Gaucher disease), glycogen storage disease type II, pompe disease caused by mutations or deletions of acid alpha-glucosidase (GAA; catalyzing degradation of glycogen) function or expression), saponin 2-I (tachese) disease, Type GM2-II gangliosidosis (Sandhoff disease), type I mucolipidosis (salivary gland disease type I and II), type II mucolipidosis (I-cell disease), type III mucolipidosis (pseudohercules disease) and type IV mucolipidosis, mucopolysaccharidosis (Hewler disease and variants thereof), Hunter (Hunter), type A, B, C, D Sanfilippo (filippo), Moquine types A and B (Morquio), Marlotte-Kaglong-Lamisy disease (Maroteax-Lamy and Sly disease), Niemann-Pick disease types A/Manman B, C1 and C2 (Niemann-Pick disease), and type I and II Scheindreader disease (vascular disease), hereditary copper disease (E), or edema (Wilken disease), such as Wilson disease or Wilson's disease, Lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), solid organ (e.g., brain, liver, kidney, heart) diseases or infectious virus (e.g., hepatitis b and c, HIV, etc.), bacterial or fungal diseases. Suitable human subjects whose biological samples can be analyzed according to the methods of the invention additionally include subjects suffering from a coagulation disorder, such as, but not limited to, subjects suffering from: hemophilia a, hemophilia B, lack of any coagulation factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor or FV/FVIII combined deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency.

Subjects whose biological samples can be analyzed using the method according to the invention also include, for example, but not limited to, those who have developed inhibitory antibodies against proteins delivered to the subject for therapeutic purposes, such as, but not limited to, subjects with pompe disease, hemophilia a or hemophilia B who are administered GAA, factor VIII and factor IX, respectively, may produce inhibitory antibodies against GAA, factor VIII and factor IX, respectively. Thus, subjects include subjects without inhibitory antibodies as well as subjects with inhibitory antibodies to proteins.

Suitable human subjects whose biological samples can be analyzed according to the methods of the invention further include subjects having an infection or a disease or neurodegenerative disease originating from the Central Nervous System (CNS), such as, but not limited to, alzheimer's disease, huntington's disease, ALS, hereditary spastic hemiplegia, primary lateral sclerosis, spinal muscular atrophy, kennedy's disease, polyglutamine repeat disease, parkinson's disease, and polyglutamine repeat disease, including, for example, but not limited to, spinocerebellar ataxia (e.g., SCA1, SCA2, SCA3, SCA6, SCA7, or SCA 17).

The present invention provides compositions, e.g., kits, that include a packaging material and one or more components therein. Kits typically include a label or package insert containing a description of the components or instructions for in vitro, in vivo, or ex vivo use of the components therein. Kits can comprise a collection of such components, e.g., a recombinant vector (e.g., rAAV) vector, an empty capsid AAV particle, and optionally one or more reagents suitable for performing the methods of the invention.

A kit refers to a physical structure that holds one or more components of the kit. The packaging material can maintain the sterility of the components and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, and the like).

The label or insert may include identification information, quantity of one or more components therein. The label or insert may include information identifying the manufacturer, lot number, manufacturing location and date, and expiration date. The label or insert may include information identifying manufacturer information, lot number, and date. The label or insert may include information regarding how to use the kit components. The label or insert may include instructions for using one or more kit components in the methods or uses of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein

All patents, patent applications, publications, and other references cited herein, GenBank citations, and ATCC citations, are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

All of the features disclosed herein may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are examples of classes of equivalent or similar features.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids, reference to "a vector" includes a plurality of such vectors, and reference to "a virus" or "particle" includes a plurality of such viruses/particles.

As used herein, the term "about" refers to a value within 10% (i.e., plus or minus 10%) of the underlying parameter. For example, "about 1: 10" means 1.1:10.1 or 0.9:9.9, about 5 hours means 4.5 hours or 5.5 hours, etc. The term "about" at the beginning of a string of values modifies each value by 10%.

All values or ranges of values include the whole number within the range and the fraction of the value or integer within the range. Thus, for example, reference to 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, also for purposes of illustration, reference to a numerical range, e.g., "1-4" includes 1, 2, 3, and 1.1, 1.2, 1.3, 1.4, etc., etc. For example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

Further, reference to a numerical range, such as "0.01 to 10" includes 0.011, 0.012, 0.013, etc., and 9.5, 9.6, 9.7, 9.8, 9.9, etc., and so forth. For example, a range of about "0.01 to about 10" includes 0.011, 0.012, 0.013, 0.014, 0.015, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc., and so forth.

References to more (greater) or less than an integer include any number greater or less than the reference number, respectively. Thus, for example, reference to more than 2 includes 2.1, 2.2, 3, 3.1, 3.2, 4, 4.1, 4.2, 5, 5.1, 5.2, etc., and so forth. Reference to "two or more times" includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.

Further, reference to numerical ranges, such as "1 to 90" includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc., and so forth. For example, "between about 1 minute and about 90 days" includes 1.0 minute, 1.1 minute, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days …, 81 days, 82 days, 83 days, 84 days, 85 days, etc.

This disclosure generally uses certain language to describe the numerous embodiments of the present invention. The invention also specifically includes embodiments in which particular subject matter, e.g., materials or materials, method steps and conditions, design or procedures, are wholly or partially excluded. For example, in certain embodiments of the present invention, materials and/or method steps are excluded. Thus, even though the invention is not generally expressed herein in terms of what is not intended to be encompassed by the invention, aspects are disclosed herein that are not expressly excluded from the invention.

Various embodiments of the present invention have been described. However, various changes and modifications can be made by one skilled in the art to adapt the invention to various usages and conditions without departing from the spirit and scope of the invention. The following examples are therefore intended to illustrate, but not limit in any way, the scope of the present invention.

Examples

Example 1

This is a description of an exemplary cell-based in vitro assay for determining titers of anti-AAV neutralizing antibodies (nabs) from serum or plasma samples using AAV vectors expressing luciferase as reporter transgenes.

The present description is applicable to determining AAV NAb titers in serum and plasma and other biological samples from human clinical trial subjects, preclinical or non-clinical studies, human candidates for gene therapy treatment methods, and monitoring AAV NAb in subjects, e.g., subjects that have received gene therapy treatment or are at risk or have developed an AAV NAb, and wherein it is desirable to determine the presence or amount of NAb before and/or after treatment, and optionally reduce the amount of AAV NAb.

An exemplary 2V6.11 cell line (Mohammadi, et al, nucleic. acids res.32:2652(2004)) used was a genetically modified Human Embryonic Kidney (HEK)293 cell line that stably expresses the E4 gene from adenovirus. E4 gene expression ensures efficient transduction of AAV vectors. Other cells are also suitable for use.

The serum sample was used as a source of potential AAV nabs, which would reduce AAV reporter vector transduction of 2V6.11 cells and decrease measured luciferase activity, if present. Serum samples are diluted, for example, but not limited to, prepared at a range of dilutions (e.g., 1:1, 1:2.5, 1:5, 1:10, 1:100, and 1:1000) and mixed with the reporter vector, so that AAV nabs (if present) bind to the AAV reporter capsid surface and neutralization occurs.

Some people, and therefore their sera (or other biological samples), may contain other factors that do not bind to AAV capsids, but that may positively affect (increase cell transduction) or negatively affect (decrease cell transduction) AAV vector cell transduction, which are referred to herein as enhancers and inhibitors, respectively. Thus, in parallel, serial dilutions of serum samples were also preincubated with empty capsid AAV particles (EV) prior to addition of AAV reporter vectors.

Preincubation of serum with empty capsid AAV particles (EV) provides a method of analyzing low titer (e.g., less than or equal to about 1:5, or ≦ 1:5) or negative NAb samples for the presence of enhancers or inhibitors, e.g., in a subject prior to administration of AAV vector. This enables the identification of false NAb positive subjects due to the presence of inhibitors and false NAb negative subjects due to the presence of enhancers. False NAb-positive subjects can be treated by AAV vector-based gene therapy methods. Depending on the NAb titer, it may be decided to recruit or exclude false NAb-negative subjects from AAV vector-based gene therapy trials or methods. Clinical samples taken from subjects two weeks after AAV vector infusion typically have high NAb titers (e.g., greater than or equal to about 1:5 or ≧ 1:5) and the use of empty capsid AAV particles (EVs) is not necessary for determination of NAb titers in these subjects.

Quality Control (QC) may also be used to verify the integrity and/or accuracy of the assay. For example, an AAV NAb control can confirm that empty capsid AAV particles (EVs) are absorbing/binding AAV nabs for use in an assay to determine the presence of any enhancer or inhibitor in a biological sample. This quality control is referred to herein as EV efficacy.

The source of AAV nabs may be provided by one or more samples from one or more subjects expressing AAV nabs. Typically, AAV nabs are from pooled samples to ensure AAV nabs are present in the control. However, any form of AAV NAb, e.g., in PBS solution, can be used as a control as disclosed herein.

2V6.11 cells were first transduced into 96-well plates with the prepared AAV reporter suspension and incubated overnight. Luciferase activity was read by a luminescence plate reader. After background (MIN) subtraction of the entire plate, the results of samples run in triplicate wells were compared to the Maximum (MAX) signal generated by positive control wells containing cells transduced with AAV reporter vector only without serum. The dilution of the serum sample at which luciferase activity is inhibited by about 50% or more is reported as AAV NAb titer of the subject providing the sample.

In an exemplary study, samples were collected from 89 human subjects prior to enrollment of a phase I/II gene therapy trial for treatment of hemophilia a. This assay showed that of the 89 subjects evaluated, 58 (65.2%) subjects had AAV NAb titers <1:1, which were considered negative; the titre was >1:1 but equal to or lower than 1:10 in 16 (18%) subjects, and >1:10 in 15 (16.8%) subjects.

Example 2

Example 3

Materials and apparatus

The cells were a 2V6.11 cell line (Mohammadi, et al. Nucl. acids Res.32:2652(2004)), which is a Human Embryonic Kidney (HEK) cell line stably expressing the adenovirus E4 gene. Master, working and test ready cell banks are generated. An aliquot of the "cell bank", 1e7/mL, was stored in liquid nitrogen.

Reagents and storage conditions AAV-80 ℃ Renilla, Promega, Cat # E2820. -20 ℃ FACT (), Cat No. H101-01, -20 ℃ DMEM (Dulbecco's modified Eagle Medium), Life technologies, Cat No. 11965-^TMF-68(100X, 10%), ThermoFisher scientific, Cat #24040032 Environment 100%, Sigma, Cat # E7023-500ml

Example 4

Preparation of reagents

The reagent volume can be scaled as desired.

50mL of heat-inactivated FBS was added to 55mL of complete DMEM (cDMEM). 5mL of 100 XPen/Strep/glutamine solution was added. Filtered through a sterile vial filter. An expiration date of 1 month was specified and stored at 4 ℃.

FACT QC stock solution: AAV NAb titers in different batches of FACT may be different, and the dilution protocol may be adjusted in a manner to achieve 50% inhibition between the two intermediate dilutions. Other control AAV nabs, such as plasma or serum, can be used.

Example (b): for batch 3696, heat-inactivated FACT samples were diluted 1:10 with heat-inactivated FBS and frozen in aliquots. Stored at < -60 ℃ and specified expiration date. Not exceeding 3 freeze-thaw cycles. HQC, MQC and LQC were diluted during assay setup using the following exemplary protocol:

table 1: example of QC dilution protocol (for FACT batch 3696)

Description of QC	Volume of FACT	Volume of FBS
			FACT 1:100(HQC)	10 parts of FACT, 1:10	90 parts of FBS
FACT 1:316(MQC)	1 part of FACT, 1:100	2.16 parts of FBS
			FACT 1:1000(LQC)	1 part of FACT, 1:316	2.16 parts of FBS

Ponasterone a: the vial was rapidly rotated and reconstituted in 100% ethanol at a concentration of 1 mg/mL. Vortexed vigorously. The storage was at-20 ℃.

Pluronic F68 in DPBS: one part of a 10% Pluronic stock was mixed with 10,000 parts DPBS using a two-layer dilution scheme. It is used on the day of preparation.

AAV-CAG-luciferase vector: after initial thawing of the stock solution, the vector was diluted with 0.001% Pluronic F68 to obtain 2X 10¹¹The stock solution concentration of vg/mL. A 50 μ L aliquot was dispensed into sterile non-stick surface tubes. Aliquots were stored at-60 ℃.

Empty capsid AAV particles (EV): after initial thawing of the manufacturing stock, the appropriate volume was aliquoted into sterile nonstick surface tubes and stored at-60 ℃. The residue was discarded after thawing the aliquots.

Test samples: after initial thawing, the serum samples were heat inactivated at 56 ℃ for 30 minutes. Aliquots of 400 μ L were prepared in sterile non-stick surface tubes and stored at-60 ℃ until use. After thawing, the unused serum can be stored and stored at-60 ℃ or can be discarded. If preserved, freeze-thaw cycles are marked on the tubes. Not exceeding 3 freeze-thaw cycles.

Serum dilution: fetal Bovine Serum (FBS) was prepared in one aliquot and stored at-20 ℃.

1 × Renilla luciferase assay reagent: the assay buffer and substrate are thawed or allowed to reach room temperature (ambient temperature). A water bath may be used. Mix well because thawing produces a density and composition gradient. The reagents can be thawed up to 5 times without significant loss of activity. To prepare the reagents, 1 volume of 100 × renilla luciferase assay substrate was added to 100 volumes of renilla luciferase assay buffer in a 50mL conical tube. Mix thoroughly through the vortex tube for 10-20 seconds. The reagent was placed in the dark. After preparation, the buffer was stable for 12 hours at ambient temperature.

Example 5

Day 1: plating 2V6.11 cells, sample neutralization, and cell transduction

The cDMEM medium was placed at 37 ℃ CO₂In the incubator, the lid is loosened for at least 30 minutes. This enables resetting of the pH and temperature. This medium was later used for cell thawing and plating.

Samples, FACT QC stock, heat-inactivated FBS and FBS-free DMEM were removed from storage and allowed to equilibrate to ambient temperature.

Optionally: if the serum sample was not heat inactivated, the sample was heat inactivated at 56 ℃ for 30 minutes. Tubes were labeled to indicate heat inactivation. If the previous heat inactivated, the sample was not heated again.

As shown in fig. 10A, dilutions were prepared from FACT QC stock using 96-well U-bottom tissue culture plates. This plate is called the dilution plate. The indicated volume is sufficient to prepare a sample, which should be scaled up if necessary. Pipette to mix until uniform. Using the same dilution plate, a sample dilution was prepared as shown in fig. 10B.

20 μ L of each diluted FACT control and sample was transferred from the dilution plate to a new 96-well U-bottom neutralization plate, as shown in FIG. 11.

20 μ L of heat-inactivated FBS was added to MIN, MAX and MAX.EV wells on the neutralization plate as shown in FIG. 12.

Working concentrations of 1.5X 10 were prepared in DMEM without FBS only when EV was used¹¹cp/mL of empty capsid AAV particles (EV). Avoiding severe vortexing. A 0.8mL volume of dilution EV is sufficient for one complete test plate.

Only when EV was used, 10 μ L of EV was added to the wells designated as s.ev, fact.ev, and max.ev, as shown in fig. 13A. 10 μ L of DMEM without FBS was added to wells containing sample (S), FACT and MAX, and 20 μ L of DMEM without FBS was added to MIN control wells as shown in FIG. 13B. When only EV was used, the neutralized plates were incubated at 37 ℃ for 30 ± 5 minutes.

Working concentration was 3.2X 10 in DMEM without FBS⁹vg/mL of AAV Reporter Vector (RV). Avoiding severe vortexing. A volume of 0.8mL of diluted vehicle was sufficient to accommodate one complete test plate.

Add 10 μ L RV to all wells except wells containing MIN control in the neutralization plate as shown in figure 14. The neutralized plates were incubated at 37 ℃ for 30. + -.5 minutes.

A vial of 2V6.11 cells was removed from the freezer and immediately placed in a 37 ℃ water bath for 4 minutes. If multiple vials are required, the corresponding increase. The vial was removed from the water bath, rinsed with 70% EtOH and inverted 180 ° twice to resuspend the cells.

All cell suspensions were aspirated from the cryovial using a1 or 2mL serum pipettor and transferred to an empty 15mL conical tube. At this step, the cells are vulnerable to shear forces.

Using a 10mL pipette, 9mL of warm medium was added to a 15mL conical tube. The first 3mL should be added slowly, dropwise. The remaining 6mL of medium was added faster from the pipette. The cells are now suspended in about 10 mL.

The 15mL conical tube was closed and inverted 180 ° twice to homogenize the cell suspension. 50 μ L of the cell suspension was transferred to an Eppendorf tube for counting, and the remaining cell suspension was spun at 240 Xg for 10 minutes.

Live and dead cells were calculated. If the viability is below 70%, the cells are discarded and another vial is thawed.

Cells were diluted to 4.0X 10 in cDMEM⁵Individual cells/mL. Ponasterone A was added to a final concentration of 1. mu.g/mL. Each plate required 10mL of cell suspension.

Flat bottom 96 well tissue culture plates were prepared and 100. mu.L of cell suspension (4.0X 10) was added to each well⁴Individual cells). This plate is called a rotating guide plate. At 37 deg.C, 5% CO₂Incubate in incubator until use. The lot number and viability of the cell banks used were recorded.

After neutralization, 7.5 μ L from each well on the neutralization plate was transferred in triplicate to wells in the transfer plate, as shown in fig. 15. Wrapping the transfer plate with sealing film and heating at 37 deg.C with 5% CO₂The transfer plate was incubated in the tissue culture incubator for 24 hours. + -. 30 minutes. The incubation start time was recorded.

Day 2: measurement of luciferase Activity

Cell culture supernatant containing secreted luciferase should be removed from the wells within about 24 to 25 hours after the added 7.5. mu.L/well is transferred from the neutralization plate to the transfer plate. Since secreted luciferase accumulates in the culture medium over time, varying this time greatly affects the range of activity readings.

Sufficient volumes of 1 × Renilla luciferase assay reagent were prepared and equilibrated to room temperature prior to assay. The test plate was removed from the incubator. Cells were observed and any signs of toxicity (low degree of fusion, cells detached from the bottom of the well, cells attached but rounded, etc.) were recorded and annotated.

The GloMax Navigator System microplate reader was turned on prior to reading, and the plate PC was then turned on for at least five minutes. When incubation was complete for about 24 hours, the supernatant was pipetted down once using a multichannel pipettor and about 90 μ Ι _ of culture supernatant was transferred to a 96-well tissue culture plate. Excess supernatant can be used for a second reading if desired. If not used, it can be discarded or frozen at < -60 ℃.

Transfer 40. mu.L of culture supernatant to a 96-well blackboard. The detector board layout is shown in fig. 7. Optionally, if the plate is not read immediately, the plate is sealed with a sealant and stored at room temperature in the dark for up to 4 hours.

The reader is loaded with a plate. Click on the GloMax Navigator software icon to launch the GloMax Navigator software. A protocol is selected. The instrument set-up detailed below was used:

a reading mode: luminescence

Integration time: 1 second

Plate type: 96 well standard

Reading the area: highlighting columns and rows to be measured

Injection and delay:

an injector 1: deselection

An injector 2: selecting

Volume: 100 μ L

Delaying: 2 seconds

Speed: 230 μ L/sec

Dark adaptation: 3 minutes

Reading board

Example 6

Data analysis

The mean (also called "mean") Standard Deviation (SD) and% CV of luminescence readings [ RLU ] were calculated for all control and sample dilutions.

Mean luminescence value RLU using MAX and MIN_AV]To calculate the signal-to-noise ratio (S/N). S/N MAX RLU_AV]/MIN[RLU_AV]

Mean luminescence values [ RLU ] using MAX, MIN, QC dilutions with and without empty vector (e.g., HQC, MQC, LQC, HQC EV, MQC EV, and LQC EV)_AV]And triplicate test sample (S) wells were used for the following calculations, respectively:

% inhibition MAX (% i.max) 100- [ (S-MIN)/(MAX-MIN) ] × 100 ]. This was calculated for each sample (S) dilution as shown in table 2.

TABLE 2

Degree of dilution	% inhibition MAX (% I.MAX)
		1:1	100-[(S 1:1-MIN)/(MAX-MIN)]×100]
1:2.5	100-[(S 1:2.5-MIN)/(MAX-MIN)]×100]
		1:5	100-[(S 1:5-MIN)/(MAX-MIN)]×100]
1:10	100-[(S 1:10-MIN)/(MAX-MIN)]×100]
		1:100	100-[(S 1:100-MIN)/(MAX-MIN)]×100]
1:1000	100-[(S 1:1000-MIN)/(MAX-MIN)]×100]

% inhibition s.ev (% i.sev) ═ 100- [ (S-MIN)/(s.ev-MIN) ] × 100 ]. This was calculated for each sample (S) dilution as shown in table 3.

TABLE 3

Degree of dilution	% inhibition S.EV (% I.SEV)
		1:1	100-[(S 1:1-MIN)/(S.EV 1:1-MIN)]×100]
1:2.5	100-[(S 1:2.5-MIN)/(S.EV 1:2.5-MIN)]×100]
		1:5	100-[(S 1:5-MIN)/(S.EV 1:5-MIN)]×100]
1:10	100-[(S 1:10-MIN)/(S.EV 1:10-MIN)]×100]
		1:100	100-[(S 1:100-MIN)/(S.EV 1:100-MIN)]×100]
1:1000	100-[(S 1:1000-MIN)/(S.EV 1:1000-MIN)]×100]

HQC% inhibition 100- [ (HQC-MIN). times.100)/(MAX-MIN) ]

EV interference is MAX/MAX

EV efficacy is HQC EV/HQC

AAV NAb titers were defined as the lowest dilution of the sample that inhibited ≧ 50. NAb titers were assigned to each sample as shown in figures 6A-6E.

Detection acceptance criteria

Detection standard 1: the HQC (e.g., FACT 1:100) must meet the predefined batch-specific% i.max ± 3 SD.

Detection standard 2: HQC (e.g., FACT 1:100) must meet the accuracy of CV% ≦ 35% for the value [ RLU ] in three replicate wells.

Detection standard 3: positive transduction controls (MAX) must meet the accuracy of CV% ≦ 35% for luminescence [ RLU ] in triplicate wells.

Detection criteria 4 (applicable only when EV is used for detection): the calculated EV interference must be in the range of 0.7 to 1.3.

Detection criteria 5 (applicable only when EV is used for detection): the calculated EV must be ≧ 2.

Test panels that do not meet all of the above applicable criteria are considered to be failure panels. No results should be reported for the failed panels. The assay was repeated.

Sample titer acceptance criteria

Standard 1: for the luminescence [ RLU ] values between the three replicate wells, the sample dilution reported as the final NAb titer should meet the accuracy of CV% ≦ 35%. For any sample that does not meet the standard, the result will be deemed invalid and will need to be retested unless the sample is exhausted or there is a clear reason. Samples with valid results should not be reanalyzed unless a technical error is found or reanalyzed is required.

Example 7

TABLE 4

Example 8

Spk1(SEQ ID NO:1):

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

Spk2(SEQ ID NO:2):

MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

CAG promoter sequence (also shown schematically in FIG. 16) (SEQ ID NO:3):

ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAA