Gene therapy for the treatment of familial hypercholesterolemia

文档序号：1894713 发布日期：2021-11-26 浏览：13次中文

阅读说明：本技术 用于治疗家族性高胆固醇血症的基因疗法 (Gene therapy for the treatment of familial hypercholesterolemia ) 是由 D·J·拉德于 2019-12-19 设计创作，主要内容包括：描述了可用于治疗患有家族性高胆固醇血症的人类患者的方案。此类方案包括共施用皮质类固醇与包括LDLR的复制缺陷型重组腺相关病毒(rAAV)的悬浮液。(Protocols useful for treating human patients with familial hypercholesterolemia are described. Such regimens include co-administering a suspension of a corticosteroid and a replication-deficient recombinant adeno-associated virus (rAAV) including LDLR.)

1.A regimen for treating Familial Hypercholesterolemia (FH), the regimen comprising administering to a patient a suspension comprising a recombinant adeno-associated virus (rAAV) viral particle comprising a vector genome packaged in an AAV8 capsid, wherein the vector genome comprises an AAV Inverted Terminal Repeat (ITR) and a nucleic acid sequence encoding a human Low Density Lipoprotein (LDL) receptor (hLDLR) operably linked to a liver-specific promoter, wherein the suspension is at about 2.5 x 10 per kilogram (kg) body weight of the patient¹³A dose of the rAAV viral particle of individual Genomic Copies (GC) is administered to the patient, and wherein the genomic copies are determined by optimized quantitative polymerase chain reaction (oqPCR) or digital droplet polymerase chain reaction (ddPCR).

2. The regimen according to claim 1, further comprising treating the patient with a steroid.

3. The regimen according to claim 1 or 2, wherein the suspension is an aqueous solution further comprising a formulation buffer comprising phosphate buffered saline.

4. The regimen according to any one of claims 1-3, wherein the suspension comprises a concentration or potency of at least 1 x 10¹³GC/ml of the rAAV viral particle.

5. The regimen according to any one of claims 1-4, wherein the rAAV viral particle is at least about 95% free of empty capsids.

6. The regimen according to any one of claims 1-5, wherein the rAAV viral particle has a ratio of empty capsid to intact particle between 0:4 and 1:4, inclusive of the endpoints and any ratio therebetween.

7. The regimen according to any one of claims 1-6, wherein 5 x 10¹¹GC/kg doses of the rAAV viral particles reduced baseline cholesterol levels in a Double Knockout (DKO) LDLR-/-Apobec-/-mouse model of homozygous familial hypercholesterolemia (HoFH) by 25% to 75%.

8. The regimen according to any one of claims 1 to 7, wherein the liver-specific promoter is a thyroxine-binding globulin (TBG) promoter.

9. The protocol of claim 8, wherein the TBG promoter is a human TBG promoter.

10. The regimen according to any one of claims 1 to 9, wherein the hLDLR nucleic acid sequence comprises the sequence of SEQ ID No. 2 or 4 or nucleotides (nt)969 to nt 3551 of SEQ ID No. 6.

11. The protocol of any one of claims 1-10, wherein the vector genome further comprises one or more of an intron, an enhancer, and a polyA signal.

12. The regimen according to claim 11, wherein the enhancer is an alpha 1 microglobulin/bichonitz inhibitor (bikunin) enhancer (alpha mic/bik) enhancer.

13. The protocol of claim 11 or 12, wherein the polyA signal is rabbit β -globin polyA.

14. The protocol of any one of claims 1 to 13, wherein the vector genome comprises the sequence of nucleotides (nt)1 to nt 3947 of SEQ ID No. 6.

15. The regimen according to any one of claims 1-14, wherein the suspension further comprises a surfactant.

16. The regimen according to claim 15, wherein the surfactant is a poloxamer.

17. The regimen according to claim 15 or 16, wherein the surfactant is present at a concentration of from about 0.0005% to about 0.001% of the suspension.

18. The regimen according to any one of claims 1-17, wherein the pH of the suspension ranges from 6.5 to 8 or from 7 to 7.5.

19. The regimen according to any one of claims 1-18, wherein the suspension further comprises a formulation buffer comprising 180mM NaCl, 10mM sodium phosphate, and 0.001% poloxamer 188, at a pH of 7.3.

20. The regimen according to any one of claims 1-19, wherein the suspension is administered to the patient by peripheral intravenous infusion.

21. The regimen according to any one of claims 1-20, wherein the patient is a human patient diagnosed with homozygous fh (hofh).

22. The regimen according to any one of claims 1-21, wherein the patient is treated with a steroid.

23. The regimen according to claim 22, wherein the patient is treated with steroid co-therapy for about 14 weeks.

24. The regimen of claim 22, wherein the steroid is administered to the patient in a decreasing dose equivalent to an initial dose of about 40 mg/day prednisone from the day prior to administration of the rAAV suspension (i.e., day-1) to about week 8 after rAAV administration.

25. The regimen according to claim 24, wherein the steroid is further administered to the patient at a 10mg dose reduction/week for each of weeks 9 and 10 and at a 5mg dose reduction/week for each of weeks 11, 12 and 13, such that the steroid stops after week 14, optionally when the patient receives about 2.5 x 10¹³GC/kg to 7.5X 10¹²The steroid regimen is also delivered at the dosage of the individual genome copies or other dosages provided herein.

26. The regimen according to any one of claims 22-25, wherein the steroid is prednisone.

27. A suspension for treating Familial Hypercholesterolemia (FH) comprising a recombinant adeno-associated virus (rAAV) viral particle comprising a vector genome packaged in an AAV8 capsid, wherein the vector genome comprises an AAV Inverted Terminal Repeat (ITR) and a nucleic acid sequence encoding a human Low Density Lipoprotein (LDL) receptor (hLDLR) operably linked to a liver-specific promoter, wherein the suspension is suitable for about 2.5 x 10 per kilogram (kg) body weight of a patient¹³A dose of the rAAV viral particle of individual Genomic Copies (GC) is administered to the patient, and wherein the genomic copies are determined by optimized quantitative polymerase chain reaction (oqPCR) or digital droplet polymerase chain reaction (ddPCR).

28. Use of the regimen according to any one of claims 1-26 or the suspension of claim 27 for treating a patient suffering from FH.

29. Use of the regimen of any one of claims 1-26 or the suspension of claim 27 in the manufacture of a medicament for treating FH.

30. The regimen of any one of claims 1-26 or the suspension of claim 27, for use in the manufacture of a medicament for the treatment of FH.

31. A method for treating a patient with Familial Hypercholesterolemia (FH), the method comprising administering to the patient a suspension comprising a recombinant adeno-associated virus (rAAV) viral particle, wherein the rAAV viral particle comprises a vector genome packaged in an AAV8 capsid, wherein the vector genome comprises an AAV Inverted Terminal Repeat (ITR) and a nucleic acid sequence encoding a human Low Density Lipoprotein (LDL) receptor (hLDLR) operably linked to a liver-specific promoter, wherein the patient is administered about 2.5 x 10 per kilogram (kg) body weight of the patient¹³(ii) individual Genomic Copies (GC) of the rAAV viral particle, and wherein the genomic copy number or titer is determined by optimized quantitative polymerase chain reaction (oqPCR) or digital droplet polymerase chain reaction (ddPCR).

32. The method of claim 31, wherein the suspension is an aqueous solution comprising the rAAV viral particle and a formulation buffer.

33. The method of claim 32, wherein the formulation buffer comprises phosphate buffered saline and a surfactant.

34. The method of any one of claims 31-33, wherein the suspension has at least 1 x 10¹³GC/ml rAAV Genome Copy (GC) titers.

35. The method of any one of claims 31-34, wherein the suspension is at least about 95% free of empty capsids.

36. The method of any one of claims 31-35, wherein the suspension has a ratio of empty capsids to intact rAAV viral particles between 0:4 and 1:4, inclusive of the endpoints and any ratio therebetween.

37. The method of any one of claims 31-36, wherein 5 x 10¹¹GC/kg doses of the rAAV viral particles reduced baseline cholesterol levels in a Double Knockout (DKO) LDLR-/-Apobec-/-mouse model of homozygous familial hypercholesterolemia (HoFH) by 25% to 75%.

38. The method of any one of claims 31-37, further comprising administering a steroid to the patient.

39. The method of any one of claims 31-38, wherein the suspension comprises a concentration or titer of at least 1 x 10¹³GC/ml of the rAAV viral particle.

40. The method of any one of claims 31 to 39, wherein the liver-specific promoter is a thyroxine-binding globulin (TBG) promoter.

41. The method of claim 40, wherein the TBG promoter is a human TBG promoter.

42. The method of any one of claims 31 to 41, wherein the hLDLR nucleic acid sequence comprises the sequence of SEQ ID NO. 2 or 4 or nucleotides (nt)969 to nt 3551 of SEQ ID NO. 6.

43. The method of any one of claims 31-42, wherein the vector genome further comprises one or more of introns, enhancers and polyA signals.

44. The method of any one of claims 31-43, wherein the enhancer is an alpha 1 microglobulin/bis Cunnitz inhibitor enhancer (alpha mic/bik) enhancer.

45. The method of any one of claims 31-44, wherein the polyA signal is rabbit β globulin polyA.

46. The method according to any one of claims 31 to 45, wherein the vector genome comprises the sequence of nucleotides (nt)1 to nt 3947 of SEQ ID NO 6.

47. The method of any one of claims 31-46, wherein the suspension or the formulation buffer further comprises a surfactant.

48. The method of claim 47, wherein the surfactant is a poloxamer.

49. The method of claim 47 or 48, wherein the surfactant is present at a concentration of about 0.0005% to about 0.001% of the composition.

50. The method of any one of claims 31-49, wherein the pH of the suspension ranges from 6.5 to 8 or from 7 to 7.5.

51. The method of any one of claims 31-50, wherein the suspension further comprises a formulation buffer comprising 180mM NaCl, 10mM sodium phosphate, and 0.001% poloxamer 188, at a pH of 7.3.

52. The method of any one of claims 31-51, wherein the suspension is administered to the patient by peripheral intravenous infusion.

53. The method of any one of claims 31-52, wherein the patient is a human patient diagnosed with homozygous FH (HoFH).

54. The method of any one of claims 31-53, wherein the patient is co-treated with an immunosuppressive agent.

55. The method of claim 54, wherein the patient is treated with steroid co-therapy for about 14 weeks.

56. The method of claim 54, wherein the immunosuppressant is administered to the patient in a decreasing dose equivalent to an initial dose of about 40 mg/day prednisone from the day prior to administration of the rAAV suspension (i.e., day-1) to about 8 weeks after administration of the rAAV.

57. The method of claim 56, wherein the patient is further administered the immunosuppressive agent at a 10mg dose reduction/week for each of weeks 9 and 10 and at a 5mg dose reduction/week for each of weeks 11, 12, and 13, such that the immunosuppressive agent stops after week 14.

58. The method of any one of claims 54-57, wherein the immunosuppressive agent is the steroid prednisone.

Technical Field

The present invention relates to a gene therapy for the treatment of Familial Hypercholesterolemia (FH), and in particular homozygous FH (hofh).

Background

Familial Hypercholesterolemia (FH) is a life-threatening condition caused by mutations in genes that affect LDL receptor (LDLR) function (Goldstein et al, "Familial hypercholesterolemia in The Metabolic and Molecular basis of Inherited diseases," edited by c.r. scanner et al, 2001, McGraw-high Information Services Company, New York (New York), pages 2863 through 2913 (2001)). It is estimated that > 90% of molecularly confirmed FH patients carry mutations in the gene encoding LDLR (LDLR, MIM 606945). The remaining patients carry mutations in three additional genes: APOB (MIM 107730) encoding apolipoprotein (apo) B, PCSK9(MIM 607786) encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLRAP1(MIM 695747) encoding LDLR adaptor protein 1. The latter is the only genetic mutation associated with the recessive trait. Homozygosity is usually conferred by mutations present in 2 alleles of the same gene; however, cases of patients with double heterozygous mutations (one in each of two different genes) have been reported. Prevalence based on heterozygous FH is between 1 in 500 and 1 in 200 (Nordestgaard et al, journal of european Heart disease (Eur Heart J.), 2013.34(45): pages 3478 to 90a (2013), Sjouke et al, journal of european Heart disease (2014)), estimating 7,000 to 43,000 people worldwide to have homozygous FH (hofh).

Characterization of mutant LDLR alleles has revealed a variety of mutations including deletions, insertions, missense mutations, and nonsense mutations (Goldstein et al 2001). More than 1700 LDLR mutations have been reported. This heterogeneity of genotypes leads to variable outcomes of the biochemical function of the receptors, which are classified in four general groups. Class 1 mutations are associated with undetectable proteins and are usually caused by gene deletions. Class 2 mutations result in aberrant intracellular processing of proteins. Class 3 mutations specifically affect binding to ligand LDL, and class 4 mutations encode receptor proteins that do not aggregate in the capsule pits. Mutations were also classified as receptor negative (< 2% residual activity of LDLR) or receptor deficient (2% to 25% residual activity) based on residual LDLR activity assessed using patient cultured fibroblasts. The mean LDL-C levels are lower in receptor deficient patients and the cardiovascular course is less malignant.

Untreated plasma total cholesterol levels in patients with HoFH are typically greater than 500mg/dl due to impaired LDL receptor function, resulting in premature and aggressive atherosclerosis, which often leads to cardiovascular disease (CVD) before the age of 20 and death before the age of 30 (Cuchel et al journal of european cardiology, 2014.35(32): pages 2146 to 2157 (2014), Goldstein et al 2001). It is therefore necessary to start active treatment for these patients as early as possible (Kolansky et al 2008). The available options are limited. Statins are considered first line drugs of drug therapy. Even with the largest dose, only 10% to 25% reductions in plasma levels of LDL-C are observed in most patients (Marais et al, "Atherosclerosis (Atherosclerosis), 2008.197(1): pages 400-6 (2008); Raal et al," Atherosclerosis, "2000.150 (2): pages 421-8 (2000)). The addition of the cholesterol absorption inhibitor ezetimibe (ezetimibe) in statin therapy may result in a further 10% to 20% decrease in LDL-C levels (Gagne et al Circulation, 2002.105(21): 2469-2475 (2002)). The use of cholesterol lowering drugs, including bile acid sequestrants, nicotinic acid, fibrates (fibrates) and probucol (probucol), has been successfully applied in the former statin era and may be considered to achieve further reductions in LDL-C in HoFH; however, the use of cholesterol-lowering drugs is limited by tolerance and drug availability. This approach has been shown to reduce CVD and all-cause mortality (Raal et al, Loop 2011.124(20): pages 2202-7). Despite the implementation of active multiple drug treatment approaches, HoFH patients maintain elevated LDL-C and their average life expectancy is maintained at about 32 years of age (Raal et al 2011). Several non-pharmaceutical options have been tested over the years. Surgical interventions such as portal vein shunts (Bilheimer atherosclerosis, 1989.9 (supplement 1): pages I158 to I163 (1989)), Forman et al atherosclerosis, 1982.41(2-3): pages 349 to 361 (1982)) and ileal shunts (Deckelbaum et al, New England journal of medicine 1977; 296: 465-4701977.296 (9): pages 465 to 470 (1977)) caused only local and temporary reductions in LDL-C and are now considered unsuccessful. Orthotopic liver transplantation has been shown to significantly reduce LDL-C levels in patients with HoFH (Ibrahim et al journal of cardiovascular transformation studies (J cardiovascular trans Res), 2012.5(3): pages 351-8 (2012); kucukkartalar et al 2 pediatric transplantation (Pediatr Transplant), 2011.15(3): pages 281-4 (2011)), but drawbacks and risks limit the use of this approach, including high risk of post-Transplant surgical complications and death, scarcity of donors, and the need for life-long treatment with immunosuppressive therapy (mallatack pediatric transplantation, 2011.15(2): pages 123-5 (2011); Lancet et al Lancet (last), 1984.1(8391): page 1382 1383 (1984)). The current standard of care for HoFH involves lipoprotein apheresis, a physical method of purifying plasma in LDL-C that can temporarily reduce LDL-C by more than 50% (Thompson "atherosclerosis", 2003.167(1): pages 1 to 13 (2003); Vella et al, Meio Clin Proc (Mayo Clin Proc); 2001.76(10): pages 1039-46 (2001)). Rapid accumulation of LDL-C in plasma after the treatment session (Eder and Rader, Current Trends of therapy (Today's Therapeutic Trends), 1996.14, pp 165-179 (1996)) requires weekly or bi-weekly apheresis. Although this procedure can delay the onset of atherosclerosis (Thompson et al lancet, 1995.345: pages 811-816; Vella et al clinical proceedings Meiao, 2001.76(10): pages 1039-46 (2001)), the procedure is laborious, expensive and not readily available. Furthermore, although the procedure is a generally well-tolerated procedure, the fact that the procedure requires frequent repetition and intravenous routes can be challenging for many HoFH patients.

Recently, three new drugs have gained FDA approval as an add-on therapy specifically directed to HoFH. Two of the three new drugs, lomitapide (lomitapide) and mipomessen (mipomensen), inhibit the assembly and secretion of apoB-containing lipoproteins, although they do so by different molecular mechanisms (Cuchel et al, new england journal of medicine, 2007.356(2): page 148-156 (2007); Raal et al, lancet, 2010.375(9719): page 998-1006 (2010)). This approach resulted in a significant reduction in LDL-C, on average by-50% with lomitapide (Cuchel et al 2013) and by-25% with milbexane (Rall et al 2010). However, their use is associated with a number of adverse events that can affect tolerance and long-term compliance, and include liver fat accumulation, the long-term effects of which have not been fully elucidated.

The third new class of drugs is part of a new class of lipid-lowering drugs, i.e. monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9) that have been shown to be effective in lowering LDL-C levels in patients with heterozygous FH, with clearly good safety profiles (Raal et al "circulation", 2012.126(20): pages 2408-17 (2012), Raal et al "lancets", 2015.385(9965): page 341-350 (2015); Stein et al "circulation", 2013.128(19): pages 2113-20 (2012)). Treatment of HoFH with 420mg PCSK9 inhibitor efuzumab (evolocumab) every 4 weeks for 12 weeks has been shown to provide a LDL-C reduction of about 30% compared to placebo (Raal et al 2015). However, the efficacy of PCSK9 inhibitors is dependent on residual LDLR activity, with no effect on patients without residual LDLR activity (Raal et al 2015; Stein et al, cycles, 2013.128(19): pages 2113-20 (2013)). Although the addition of PCSK9 inhibitors may become the standard of care for FH and may further reduce hypercholesterolemia in a subpopulation of HoFH patients, they do not significantly affect the clinical management of this condition.

Thus, the medical need for new drug therapies for HoFH remains unmet.

Disclosure of Invention

A protocol is provided that includes a replication-deficient adeno-associated virus (AAV) for delivering a human low density lipoprotein receptor (hLDLR) gene to liver cells of a patient (human subject) diagnosed with HoFH. A recombinant AAV vector (rAAV) for delivery of an LDLR gene ("rAAV. hLDLR") should have tropism for the liver (e.g., a rAAV with AAV8 capsid), and the hLDLR transgene should be under the control of liver-specific expression control elements. The hldlr vector may be administered by Intravenous (IV) infusion over a period of 20 to 30 minutes to reach LDLR tables in the liverTo achieve a therapeutic level. In certain embodiments, the regimen comprises administration of about 2.5 x 10¹³Hldlr range per Genome Copy (GC)/kg raav. In certain embodiments, the regimen comprises co-administering a gradually decreasing dose of a steroid (e.g., prednisone (or a steroid equivalent) equivalent to an initial dose of about 40 mg/day). In certain embodiments, treatment begins on day-1 and continues until about week 8 after dosing. In certain embodiments, the dose is reduced at a dose of 10 mg/week for each week in weeks 9 and 10, and 5 mg/week for each week in weeks 11, 12 and 13. In certain embodiments, when the patient receives about 2.5 x 10¹³GC/kg to 7.5X 10¹²Steroid regimens are also delivered at the doses of individual genomic copies or other doses provided herein.

A protocol is provided that includes a replication-deficient adeno-associated virus (AAV) for delivering a human low density lipoprotein receptor (hLDLR) gene to liver cells of a patient (human subject) diagnosed with HoFH. A recombinant AAV vector (rAAV) for delivery of an LDLR gene ("rAAV. hLDLR") should have tropism for the liver (e.g., a rAAV with AAV8 capsid), and the hLDLR transgene should be under the control of liver-specific expression control elements. The hldlr vector may be administered by Intravenous (IV) infusion over a period of 20 to 30 minutes to achieve therapeutic levels of LDLR expression in the liver. In certain embodiments, the regimen comprises administration of about 2.5 x 10¹³Hldlr range per Genome Copy (GC)/kg raav. In certain embodiments, the regimen comprises co-administering a gradually decreasing dose of a steroid (e.g., prednisone (or a steroid equivalent) equivalent to an initial dose of about 40 mg/day). In certain embodiments, prophylactic co-treatment with steroids begins at least one day prior to gene therapy (day-1) or on the day of gene therapy delivery (day 0) and continues until about week 8 after dosing. In certain embodiments, the prophylactic co-treatment begins at least one day prior to or on the same day as gene therapy delivery and continues at a gradually decreasing dose to about week 13 after administration. Optionally, prophylacticSteroid co-therapy may be initiated 2 or 3 days prior to vehicle administration. In certain embodiments, the dose is reduced at a dose of 10 mg/week for each week in weeks 9 and 10, and 5 mg/week for each week in weeks 11, 12 and 13. In certain embodiments, when the patient receives a lower dose (e.g., about 2.5 x 10)¹²GC/kg to 7.5X 10¹²GC/kg) or higher doses as provided herein, the prophylactic steroid regimen is also delivered.

The goal of such treatment is to functionally replace the patient's defective LDLR by rAAV-based liver-targeted gene therapy as a viable approach to treating this disease and improving response to current lipid-lowering therapies. The present invention is based, in part, on the development of therapeutic compositions and methods that allow for the safe delivery of effective doses; and improved manufacturing processes for meeting the purification manufacturing requirements for effective administration to human subjects.

The efficacy of the therapy can be assessed post-treatment (e.g., post-administration) using plasma LDL-C levels as a surrogate biomarker for human LDLR transgene expression in the patient. For example, a decrease in the patient's plasma LDL-C level following the gene therapy indicates successful transduction of functional LDLR. Additionally or alternatively, other parameters that may be monitored include, but are not limited to, measuring changes in Total Cholesterol (TC), non-high density lipoprotein cholesterol (non-HDL-C), HDL-C, fasting Triglycerides (TG), very low density lipoprotein cholesterol (VLDL-C), lipoprotein (a) (lp (a)), apolipoprotein b (apob), and apolipoprotein a-I (apoA-I) from baseline, as well as LDL kinetics studies (metabolic mechanism assessment) before and after vehicle administration, or combinations thereof.

In certain embodiments, the efficacy of a therapy may be measured by a reduction in the frequency of apheresis required by the patient. In certain embodiments, the patient's need for apheresis may be reduced by 25%, 50%, or more following aav8.hldlr treatment. For example, a patient receiving weekly apheresis prior to aav8.hldlr therapy may only need biweekly or monthly apheresis; in other embodiments, even less frequency of apheresis may be required, or the need may be eliminated.

In certain embodiments, the efficacy of a therapy can be measured by reducing the dose of PCSK9 inhibitor required or by eliminating the need for such therapy in patients following aav8.hldlr treatment. In certain embodiments, the efficacy of the therapy is measured by reducing the dose of statin or bile chelator required.

Patients that are candidates for treatment are preferably adults (male or female aged 18 years ≧ 18) who are diagnosed as having HoFH carrying two LDLR gene mutations; that is, patients with molecularly defined LDLR mutations at both alleles in the context of a clinical manifestation consistent with HoFH, which may include untreated LDL-C levels (e.g., LDL-C levels >300mg/dl), treated LDL-C levels (e.g., LDL-C levels <300mg/dl), and/or total plasma cholesterol levels greater than 500mg/dl, as well as premature and aggressive atherosclerosis. Treatment candidates include HoFH patients undergoing treatment with lipid lowering drugs, such as statins, ezetimibe, bile acid sequestrants, PCSK9 inhibitors, and LDL and/or plasma apheresis.

Prior to treatment, the HoFH patients should be evaluated for neutralizing antibodies (nabs) against AAV serotypes for delivering the hLDLR gene. Such nabs interfere with transduction efficiency and reduce therapeutic efficacy. HoFH patients with baseline serum NAb titers of ≤ 1:10 are good candidates for treatment using rAAV.hLDLR gene therapy protocols. However, patients with other baseline levels may be selected. Treatment of HoFH patients with serum NAb titers >1:5 may require combination therapy, such as transient co-treatment with an immunosuppressive agent prior to and/or during treatment with raav. Additionally or alternatively, the patient is monitored for liver enzyme elevation that may be treated with temporary immunosuppressant therapy (e.g., if at least about 2-fold baseline levels of aspartate Aminotransferase (AST) or alanine Aminotransferase (ALT) are observed). Immunosuppressive agents for such co-therapies include, but are not limited to, steroids, antimetabolites, T cell inhibitors, and alkylating agents.

The present invention is achieved by describing the aav8.ldlr treatment regimen for human subjects (section 6, example 1); preclinical animal data demonstrating efficacy of the treatment in animal models of disease (section 7, example 2); manufacture and formulation of therapeutic aav.hldlr compositions (sections 8.1 to 8.3, example 3); and examples of methods for characterizing the AAV vectors (section 8.4, example 3).

3.1 definition of

As used herein, "AAV 8 capsid" refers to a capsid having GenBank accession numbers: the AAV8 capsid of YP _077180 (which is incorporated herein by reference) encoding the amino acid sequence and reproduced in SEQ ID NO: 5. The invention encompasses certain variants from such encoded sequences that may comprise a sequence that is identical to GenBank accession No.: YP _ 077180; U.S. Pat. nos. 7,282,199, 7,790,449; 8,319,480, respectively; 8,962,330, respectively; the reference amino acid sequence in US 8,962,332 has a sequence with about 99% homology (i.e., less than about 1% of the variants from the reference sequence). In another embodiment, the AAV8 capsid may have the VP1 sequence of the AAV8 variant described in WO2014/124282 or the dj sequence described in US 2013/0059732 a1 or US 7588772B 2, which are incorporated herein by reference. Methods of producing capsids, their coding sequences, and methods of producing rAAV viral vectors have been described. See, for example, Gao et al, Proc. Natl. Acad. Sci. U.S. A. (100) (10),6081-6086(2003), US 2013/0045186A1 and WO 2014/124282.

As used herein, the term "NAb titer" refers to a measure of how much neutralizing antibody (e.g., anti-AAV NAb) is produced that neutralizes the physiological role of the epitope (e.g., AAV) it targets. anti-AAV NAb titers can be measured as described in Calcedo, R et al, "world-wide epidemic of Neutralizing Antibodies to Adeno-Associated Viruses" Journal of Infectious Diseases, "2009.199 (3): page 381-390, which is incorporated herein by reference.

In the context of amino acid sequences, the terms "percent (%) identity", "sequence identity", "percent sequence identity", or "percent identical" refer to residues in two sequences that are the same when aligned for correspondence. The percent identity of the amino acid sequence over the full length of the protein, the polypeptide, about 32 amino acids, about 330 amino acids or peptide fragments thereof, or corresponding nucleic acid sequence-encoding sequence generator can be readily determined. Suitable amino acid fragments can be at least about 8 amino acids in length and can be up to about 700 amino acids in length. In general, when referring to "identity", "homology" or "similarity" between two different sequences, reference is made to "aligning" the sequences to determine "identity", "homology" or "similarity". "aligned" sequences or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that typically contain corrections for missing or additional bases or amino acids compared to a reference sequence. The alignment is performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used with default settings, although those skilled in the art can change these settings as desired. Alternatively, one skilled in the art may utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithm or program. See, e.g., J.D. Thomson et al, "nucleic acids research (nucleic acids. Res.)," comprehensive comparison of multiple sequence alignments (A comprehensive comparison of multiple sequence alignments) ", 27(13): 2682-.

As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with a gene of interest and expression control sequences that function in trans or remotely to control the gene of interest.

"replication-defective virus" or "viral vector" refers to a synthetic or artificial virion in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, wherein any viral genomic sequence that is also packaged in the viral capsid or envelope is replication-defective; that is, the synthetic or artificial viral particle is incapable of producing progeny virus but retains the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gut-free" -containing only the transgene of interest, which flank the signals required for amplification and packaging of the artificial genome), but these genes can be supplied during production. This is therefore considered safe for gene therapy, since replication and infection by progeny virions will not occur unless viral enzymes required for replication are present.

It should be noted that the terms "a" or "an" mean one or more. As such, the terms "a" or "an", "one or more", and "at least one" are used interchangeably herein.

The words "comprise", "comprises" and "comprising" are to be construed as inclusive and not exclusive. The words "consisting of … … (continst)", "consisting of … … (continuations)" and variations thereof are to be construed as exclusive and not inclusive. Although the various embodiments in this specification have been presented using the language "comprising," in other instances, it is intended that the related embodiments be interpreted and described using language "consisting of … … or" consisting essentially of … ….

As used herein, unless otherwise specified, the term "about" means 10% variability relative to a given reference.

Unless otherwise defined in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and with reference to the disclosure, which provides one of ordinary skill in the art with a general guide to many terms used in this application.

Drawings

Fig. 1A-1h. effect of pre-existing AAV8 NAb on EGFP expression levels in the liver of macaques. The macaque of different types and ages is injected by peripheral vein with 3X 10¹²GC/kg of AAV8.TBG. EGFP and 7 days laterThe macaques were then sacrificed and the hepatocyte transduction of the macaques was analyzed in several ways. FIGS. 1A-1E are graphs showing neutralizing antibodies (respectively, each: AAV8) from pre-existing antibodies with different levels against AAV8<Photomicrographs of representative sections of the liver of animals 1:5, 1:10, and 1: 20). Figure 1F shows a quantitative morphometric analysis of transduction efficiency based on the percent transduction of hepatocytes. Fig. 1G shows a quantitative morphometric analysis of transduction efficiency based on relative EGFP intensity. Fig. 1H shows quantification of EGFP protein in liver lysates by ELISA. Adult cynomolgus monkeys (n-8, filled circles), adult rhesus monkeys (n-8, open triangles), and juvenile rhesus monkeys (n-5, open squares).

FIG. 2 Long-term expression of mLDLR in DKO mice. DKO mice dosed with 10¹¹GC/mouse (5X 10)¹²GC/kg) of aav8.tbg. mldlr (n ═ 10) or aav8.tbg. nlacz (n ═ 10). Cholesterol levels in serum were monitored periodically. As early as day 7 (p)<0.001) that a statistically significant difference between the two groups has been achieved and is maintained throughout the experiment. Mice were sacrificed on day 180 after vehicle administration.

Fig. 3A-3l.aav8.tbg.mldlr followed by regression of atherosclerosis in DKO mice. FIG. 3A is a set of three panels with Enface Sudan IV staining. Mouse aorta was fixed and stained with sudan IV, which stains neutral lipids. Shown are the results from 10 days 60 (day 120 of high fat diet) after vehicle administration¹¹GC/mouse AAV8.nLacZ (5X 10)¹²GC/kg) (middle), 10¹¹GC/mouse AAV8.TBG. mLDLR (5X 10)¹²GC/kg) (right) or representative aorta of animals at baseline (day 60 of high fat diet) (left). Fig. 3B is a bar graph showing the results of morphometric analysis quantifying the percentage of aorta stained with oil red O along the entire length of the aorta. Figures 3C-3K show the aortic root of these mice stained with oil red O. Fig. 3L is a bar graph showing total aortic surface percentage sudan IV staining determined at baseline (n 10), aav.tbg.nlacz (n 9), and aav8.tbg.mldlr (n 10). Oil red O damage was quantified. And (3) carrying out one-way variance analysis on the data of the atherosclerotic lesion area. By usingDunnett's test compares the experimental group with the baseline group. Repeated measures anova was used to compare cholesterol levels over time after gene transfer in different groups of mice. The statistical significance of all comparisons was designated as P, 0.05. The graph represents the mean SD value. P<0.05，**p<0.01，

Fig. 4 cholesterol levels in DKO mice injected with test or control. DKO mice were injected intravenously with 7.5X 10¹¹GC/kg、7.5×10¹²GC/kg or 6.0X 10¹³GC/kg of AAV8.TBG. mLDLR or 6.0X 10¹³GC/kg of AAV8.TBG. hLDLR or vehicle control (100. mu.l PBS). Cholesterol levels are expressed as mean ± SEM. From the same necropsy time points, each group exhibited a statistically significant reduction in serum cholesterol relative to the PBS control.

Fig. 5A-5b cholesterol levels in DKO mice injected with test article. Figure 5A shows cholesterol levels (mg/mL) in mice treated with different doses of vehicle measured on days 0, 7, and 30. Values are expressed as mean ± SEM. P < 0.05.

Figure 6A-6c. peripheral T cell response in vector injected rhesus monkeys. The presented data show the time course of T cell responses and AST levels for cynomolgus monkey 19498 (FIG. 6A), 090-0287 (FIG. 6B) and 090-0263 (FIG. 6C). For each study day, T cell responses to no stimulation, AAV8, and hLDLR measured in Spot Forming Units (SFU) per million PBMC are plotted from left to right in each graph. Macaques 19498 and 090-0287 gave positive peripheral T cell responses to the hLDLR transgene, whereas 090-0263 did not. Indicates a positive capsid reaction significantly above background.

Figure 7 schematic representation of aav8.tbg. hldlr vector.

AAV cis plasmid constructs. A) Linear representation of parental cis-cloning plasmid penn.aav.tbg.pi containing a liver-specific TBG promoter flanked by AAV2 ITR elements and a chimeric intron. B) Linear representation of the human LDLR cis plasmid penn.aav.tbg.pi.hldlr.rbg.kanr, wherein the human LDLR cDNA was cloned into penn.aav.tbg.pi between the intron and the polyA signal and the ampicillin resistance gene was replaced by the kanamycin resistance gene.

Aav trans plasmid fig. 9A-9b. FIG. 9A is a linear representation of the AAV8 trans-packaging plasmid p5E18-VD2/8 with an ampicillin resistance gene. FIG. 9B is a linear representation of the AAV8 trans-packaging plasmid pAAV2/8 with a kanamycin resistance gene.

FIGS. 10A-10B. adenovirus helper plasmids. Figure 10A shows ad helper plasmid padΔ F6 derived from parental plasmid pBHG10 through intermediates padΔ F1 and padΔ F5. FIG. 10B is a linear representation of the ampicillin resistance gene in pAd Δ F6 replaced with the kanamycin resistance gene to produce pAd Δ F6 (Kan).

Fig. 11A-11b. show a flow chart of the aav8.tbg. hldlr carrier manufacturing process.

Detailed Description

Replication-deficient rAAV is used to deliver the hLDLR gene to hepatocytes of patients diagnosed with HoFH (human subjects). hLDLR vectors should have tropism for liver (e.g., rAAV with AAV8 capsid), and hLDLR transgenes should be under the control of liver-specific expression control elements.

The hldlr vector may be administered by Intravenous (IV) infusion over a period of about 20 to about 30 minutes to achieve therapeutic levels of LDLR expression in the liver. In other embodiments, shorter (e.g., 10 to 20 minutes) or longer (e.g., greater than 30 to 60 minutes, an intermediate time, such as about 45 minutes or longer) may be selected. A therapeutically effective dose range of hLDLR of at least about 2.5 x 10¹²To 7.5X 10¹²Individual Genomic Copies (GC)/kg patient body weight. In a preferred embodiment, the potency of the rAAV suspension is such that 5 x 10 of a double knockout LDLR-/-Apobec-/-mouse model administered to HoFH (DKO mice)¹¹The GC/kg dose reduced the baseline cholesterol level by 25% to 75%. Low density lipoprotein cholesterol (LDL-C) levels can be used as a surrogate for transgene expression to assess therapeutic efficacy. The primary efficacy assessment comprises LDL-C levels 1 to 3 months (e.g., week 12) after treatment, with sustained effect for at least about 1 year (about 52 weeks) thereafter. Can measure the long-term safety of transgene expression after treatment andand (4) durability.

In certain embodiments, the efficacy of a therapy may be measured by a reduction in the frequency of apheresis required by the patient. In certain embodiments, the patient's need for apheresis may be reduced by 25%, 50%, or more following aav8.hldlr treatment. For example, a patient receiving weekly apheresis prior to aav8.hldlr therapy may only require biweekly or monthly apheresis; in other embodiments, the frequency of the need for apheresis may be even lower or may eliminate the need.

Prior to treatment, the HoFH patients should be evaluated for neutralizing antibodies (nabs) against AAV serotypes for delivering the hLDLR gene. Such nabs interfere with transduction efficiency and reduce therapeutic efficacy. HoFH patients with baseline serum NAb titers of ≤ 1:10 are good candidates for treatment with rAAV.hLDLR gene therapy regimens. Treatment of HoFH patients with serum NAb titers >1:5 may require combination therapy, such as temporary co-treatment with immunosuppressive agents before/during treatment with raav. Additionally or alternatively, the patient is monitored for liver enzyme elevation that can be treated with temporary immunosuppressant therapy (e.g., if at least about a 2-fold baseline level of aspartate Aminotransferase (AST) or alanine Aminotransferase (ALT) is observed).

In certain embodiments, such therapies may involve co-administration of two or more immunosuppressive drugs (e.g., prednisone, Mycophenolate Mofetil (MMF), and/or sirolimus (i.e., rapamycin)) within the same day. One or more of these drugs may continue to be used at the same dose or at an adjusted dose after gene therapy administration. Such therapy may last for about 1 week (7 days), about 60 days, or longer, as desired. In certain embodiments, a tacrolimus-free regimen is selected. Additional immunosuppressive co-therapies are used. Immunosuppressive agents for such co-therapies include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active on immunophilins. Immunosuppressants may comprise nitrogen mustards (nitrogen mustards), nitrosoureas (nitrosourea), platinum compounds, methotrexate (methotrexate), azathioprine (azathioprine), mercaptopurine (mercaptoprine), fluorouracil (fluorouracil), dactinomycin (dactinomycin), anthracyclines (anthracyclines), mitomycins C (mitomycin C), bleomycin (bleomycin), mithramycins (mithramycin), IL-2 receptor (CD25) or CD 3-directed antibodies, anti-IL-2 antibodies, cyclosporines (ciclosporin), tacrolimus (tacrolimus), sirolimus (sirolimus), IFN- β, IFN- γ, opioids or TNF- α (tumor necrosis factor- α) binding agents. In certain embodiments, prior to gene therapy administration, immunosuppressive therapy can begin 0, 1, 2, 7, or more days prior to gene therapy administration, or 0, 1, 2,3, 7, or more days after gene therapy administration.

Immunosuppressive agents for such co-therapies include, but are not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapamycin analogs), and cytostatic agents, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active on immunophilins. Immunosuppressants may comprise nitrogen mustards (nitrogen mustards), nitrosoureas (nitrosourea), platinum compounds, methotrexate (methotrexate), azathioprine (azathioprine), mercaptopurine (mercaptoprine), fluorouracil (fluorouracil), dactinomycin (dactinomycin), anthracyclines (anthracyclines), mitomycins C (mitomycin C), bleomycin (bleomycin), mithramycins (mithramycin), IL-2 receptor (CD25) or CD 3-directed antibodies, anti-IL-2 antibodies, cyclosporines (ciclosporin), tacrolimus (tacrolimus), sirolimus (sirolimus), IFN- β, IFN- γ, opioids or TNF- α (tumor necrosis factor- α) binding agents. In certain embodiments, immunosuppressive therapy can be initiated 0, 1, 2, 7, or more days prior to gene therapy administration, or 0, 1, 2,3, 7, or more days after gene therapy administration. Such therapies may involve co-administration of two or more drugs (e.g., prednisone, Mycophenolate Mofetil (MMF), and/or sirolimus (i.e., rapamycin)) within the same day. One or more of these drugs may continue to be used at the same dose or at an adjusted dose after gene therapy administration. Such therapy may last for about 1 week (7 days), about 60 days, or longer, as desired. In certain embodiments, a tacrolimus-free regimen is selected.

5.1 Gene therapy vectors

hLDLR vectors should have tropism for liver (e.g., rAAV with AAV8 capsid), and hLDLR transgenes should be under the control of liver-specific expression control elements. The vector is formulated in a buffer/carrier suitable for infusion into human subjects. The buffer/carrier should contain components that prevent the rAAV from adhering to the infusion tubing but do not interfere with the binding activity of the rAAV in vivo.

rAAV.hLDLR vector

Any of a number of rAAV vectors with hepatic tropism may be used. Examples of AAV that can be selected as a rAAV capsid source include, for example, rh10, AAVrh64R1, AAVrh64R2, rh8[ see, e.g., U.S. published patent application nos. 2007 and 0036760-a 1; U.S. published patent application No. 2009-0197338-A1; EP 1310571 ]. See also WO 2003/042397(AAV7 and other simian AAV), US 7790449 and US 7282199(AAV8), WO 2005/033321 and US7,906,111 (AAV9), WO 2006/110689 and WO 2003/042397(rh10), AAV 3B; US 2010/0047174 (AAV-DJ).

The hLDLR transgene may comprise, but is not limited to, one or more of the sequences provided by SEQ ID No. 1, SEQ ID No. 2 and/or SEQ ID No. 4, which are provided in the accompanying sequence listing and incorporated herein by reference. With respect to SEQ ID NO:1, these sequences comprise a signal sequence located at about base pairs 188 to about base pairs 250, and the mature protein of variant 1 spans about base pairs 251 to about base pairs 2770. SEQ ID No. 1 may also identify exons, at least one of which is not present in known alternative splice variants of hLDLR. Additionally or optionally, sequences encoding one or more of the other hLDLR isoforms may be selected. See, e.g., isoforms 2,3, 4, 5, and 6, whose sequences are available, e.g., from uniprot. For example, a common variant lacks exon 4 of SEQ ID NO:1 ((bp (255) · (377)) or exon 12(bp (1546) · (1773)). optionally, the transgene may comprise a coding sequence for a mature protein having a heterologous signal sequence SEQ ID NO:2 provides a cDNA of human LDLR and a translated protein (SEQ ID NO:3 SEQ ID NO:4 provides an engineered cDNA of human LDLR alternatively or additionally, the amino acid sequence may be translated back into a nucleic acid coding sequence, comprising both RNA and/or cDNA using web-based or commercially available computer programs and service-based companies, see e.g., reverse translated sequences by embos, ebi.ac.ac.uk/Tools/st/; Gene Infinity.

In the specific example described in the examples below, the gene therapy vector is an AAV8 vector expressing the hLDLR transgene under the control of a liver-specific promoter (thyroxine-binding globulin, TBG), designated raav8.TBG. hLDLR (see figure 6). The external AAV vector component is an icosahedral capsid of serotype 8, T ═ 1, consisting of 60 copies of the three AAV viral proteins VP1, VP2, and VP3 in a ratio of 1:1: 18. The capsid contains a single-stranded DNA rAAV vector genome.

The raav8.tbg. hLDLR genome contains the hLDLR transgene flanked by two AAV Inverted Terminal Repeats (ITRs). The hLDLR transgene comprises an enhancer, a promoter, an intron, a hLDLR coding sequence and a polyadenylation (polyA) signal. ITRs are the genetic elements responsible for replication and packaging of the genome during vector production and are the only viral cis-elements required for rAAV production. Expression of the hLDLR coding sequence was driven from a hepatocyte-specific TBG promoter. Two copies of the α 1 microglobulin/bis kunitz inhibitor enhancer element are located before the TBG promoter to stimulate promoter activity. Chimeric introns were present to further enhance expression, and a rabbit β -globin polyadenylation (polyA) signal was included to mediate termination of hLDLR mRNA transcripts.

The illustrative plasmids and vectors described herein use the liver-specific promoter thyroxine-binding globulin (TBG). Alternatively, other Liver-Specific promoters can be used [ see, for example, The Liver Specific Gene Promoter Database (The Liver Specific Gene Promoter Database), Spring Harbor laboratory (Cold Spring Harbor),http://rulai.schl.edu/LSPDalpha-1 antitrypsin (A1 AT); human albumin, Miyatake et al, J.Virol., 71: 512432 (1997), humaplb; and the hepatitis B virus core promoter, Sandig et al, "Gene therapy (Gene Ther.), 3: 10029 (1996)]. TTR minimal enhancer/promoter, alpha-antitrypsin promoter, LSP (845nt)25 (intron-free scAAV is required). Although less desirable, vectors such as viral promoters, constitutive promoters, regulatable promoters may be used in the vectors described herein [ see, e.g., WO 2011/126808 and WO 2013/04943]Or other promoters responsive to physiological cues and the like.

In addition to the promoter, the expression cassette and/or vector may contain other suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, where desired, a sequence that enhances secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, for example, the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, the LSP (TH binding globulin promoter/alpha 1 microglobulin/double Cunnitz inhibitor enhancer), and other enhancers.

These control sequences are "operably linked" to the hLDLR gene sequence. The expression cassette can be engineered onto a plasmid for the production of viral vectors. The minimal sequences required to package the expression cassette into an AAV viral particle are AAV 5 'and 3' ITRs of the same AAV origin as the capsid or of a different AAV origin (to create an AAV pseudotype). In one embodiment, the ITR sequence from AAV2 or a deleted version thereof (Δ ITR) is used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may be selected. In the case where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, the expression cassette of the AAV vector comprises AAV 5'ITR, the hLDLR coding sequence and any regulatory sequences as well as AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, called a Δ ITR, has been described in which the D sequence and terminal resolution site (trs) are deleted. In other embodiments, full-length AAV 5 'and 3' ITRs are used.

The abbreviation "sc" means self-complementary. "self-complementary AAV" refers to a plasmid or vector having an expression cassette in which the coding region carried by the recombinant AAV nucleic acid sequence has been designed to form an intramolecular double-stranded DNA template. After infection, no cell-mediated second strand synthesis is awaited, but rather the two complementary half scAAV will associate to form one double stranded dna (dsdna) that is ready for immediate replication and transcription. See, e.g., D M McCarty et al, "Self-complementing recombinant adeno-associated virus (scAAV) vectors promote efficient transduction of DNA synthesis independent of DNA synthesis," Gene therapy (8.2001), Vol.8, No. 16, p.1248 and 1254. Self-complementary AAVs are described, for example, in U.S. patent nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated herein by reference in its entirety.

5.1.2rAAV.hLDLR formulations

An hldlr formulation is a suspension containing an effective amount of a raav.hldlr vector suspended in an aqueous solution containing buffered saline, a surfactant, and a physiologically compatible salt or a salt mixture adjusted to an ionic strength equivalent to about 100mM sodium chloride (NaCl) to about 250mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration. In one embodiment, the formulation may contain, for example, about 1.5 x 10¹¹GC/kg to about 6X 10¹³GC/kg or about 1X 10¹²To about 1.25X 10¹³GC/kg as measured by, for example, in m.lock et al, "human Gene therapy (Hum Gene their Methods), 2014, 4 months; 25(2) 115-25.doi 10.1089/hgtb.2013.131.Epub 2014, 2, 14, as measured by optimized qPCR (oqPCR) or digital droplet PCR (ddPCR), which are incorporated herein by reference. For example, the suspensions provided herein can contain NaCl and KCl. The pH may be in the range of 6.5 to 8 or 7 to 7.5. Suitable surfactants and combinations thereof may be selected from poloxamers, i.e. non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly (ethylene oxide)), SOLUTOL HS 15 (polyethylene glycol-15 hydroxystearate), LABRASOL (glyceryl polyoxyoctoate), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol. In one embodiment, the formulation contains a poloxamer. These copolymers are usually designated by the letter "P" (for poloxamers) followed by three numbers: the first two digits x 100 give the approximate molecular weight of the polyoxypropylene core and the last digit x 10 gives the percentage of polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant may be present in an amount up to about 0.0005% to about 0.001% of the suspension. In one embodiment, the raav.hldlr formulation is a pharmaceutical composition comprising at least 1 x 10¹³Individual Genomic Copies (GC)/mL or more, as by, for example, in m.lock et al, human basisCause therapy, 4 months 2014; 25(2) 115-25.doi 10.1089/hgtb.2013.131.epub 2014, 14 days 2, oqPCR or digital microdroplet pcr (ddpcr), which are incorporated herein by reference. In some embodiments, the carrier is suspended in an aqueous solution at pH 7.3 containing 180mM sodium chloride, 10mM sodium phosphate, 0.001% poloxamer 188. The formulations are suitable for use in human subjects and are administered intravenously. In one embodiment, the formulation is delivered by infusion via a peripheral vein within 20 minutes (± 5 minutes). However, this time can be adjusted as needed or desired.

Hldlr, empty capsids are separated from the vector particles during the vector purification process, for example, using cesium chloride gradient ultracentrifugation, discussed in detail in section 8.3.2.5. In one embodiment, vector particles containing a packaged genome are purified from empty capsids using the process described in international patent application No. PCT/US16/65976, filed on 9/12/2016, U.S. patent application No. 62/322,093, filed on 13/4/2016, 2015, and U.S. patent application No. 62/266,341, filed on 11/12/2015, entitled Scalable Purification Method for AAV8 for AAV8, which is incorporated herein by reference. Briefly, a two-step purification scheme is described that selectively captures and isolates genome-containing rAAV vector particles from a clarified, concentrated supernatant of a rAAV-producing cell culture. . The process utilizes an affinity capture method performed at high salt concentrations, followed by an anion exchange resin method performed at high pH, to produce rAAV vector particles substantially free of rAAV intermediates. In certain embodiments, the methods isolate AAV8 viral particles comprising DNA comprising a pharmacologically active genomic sequence from a genome-deficient (empty) AAV8 capsid intermediate. The method involves: (a) forming a loading suspension comprising: recombinant AAV8 viral particles and empty AAV8 capsid intermediates purified to remove non-AAV material from the culture of AAV producer cells in which the particles and intermediates are produced; and a buffer a comprising 20 mbis-Tris propane (BTP) and having a pH of about 10.2; (b) loading the suspension of (a) onto a strong anion exchange resin, said resin being located in a container having an inlet for flow of suspension and/or solution and an outlet for flow of eluate from said container; (c) washing the loaded anion exchange resin with 1% buffer B comprising 10mM NaCl and 20mM BTP, wherein the pH is about 10.2; (d) applying an increasing salt concentration gradient to the loaded and washed anion exchange resin, wherein the salt gradient ranges from 10mM to about 190mM NaCl (inclusive) or an equivalent range; and (e) collecting the rAAV particles from the eluate, the rAAV particles being purified from the intermediate.

In one embodiment, the pH used is 10 to 10.4 (about 10.2) and the rAAV particles are purified from AAV8 intermediate at least about 50% to about 90%, or pH 10.2 and purified from AAV8 intermediate at least about 90% to about 99%. In one embodiment, this is determined by genomic copy. A stock or formulation of rAAV8 particles (packaged genome) is "substantially free" of AAV empty capsids (and other intermediates) when rAAV8 particles in the stock solution comprise at least about 75% to about 100%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of rAAV8 in the stock solution and "empty capsids" comprise less than about 1%, less than about 5%, less than about 10%, less than about 15% of rAAV8 in the stock solution or formulation.

In one embodiment, the formulation is characterized by a rAAV stock solution having a ratio of "empty" to "intact" of 1 or less (preferably less than 0.75, more preferably 0.5, preferably less than 0.3).

In further embodiments, the average yield of rAAV particles is at least about 70%. This can be calculated by determining the titer (genomic copy) in the mixture loaded onto the column and the amount present in the final elution. Further, these may be determined according to q-PCR analysis as described herein and/or SDS-PAGE techniques or those already described in the art.

For example, to calculate the content of empty and intact particles, the VP3 belt area of selected samples (e.g., formulations purified over a gradient of iodixanol (iodixanol), where GC # -particle #) was plotted against the loaded GC particles. The resulting linear equation (y-mx + c) is used to calculate the number of particles in the banded volume of the test peak. The number of particles loaded per 20 μ L (pt) was then multiplied by 50 to give particles (pt)/mL. Dividing Pt/mL by GC/mL gives the ratio of particle to genome copy (Pt/GC). Pt/mL-GC/mL gave empty Pt/mL. Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.

Generally, methods for assaying empty capsids and AAV vector particles having packaged genomes are known in the art. See, e.g., Grimm et al, Gene therapy (1999)6: 1322-1330; sommer et al, molecular therapy (molecular. Ther.) (2003)7: 122-. To test for denatured capsids, the method comprises subjecting the treated AAV stock to SDS-polyacrylamide gel electrophoresis (consisting of any gel capable of separating the three capsid proteins, e.g., a gradient gel containing 3-8% triacetate in buffer), then running the gel until the sample material is separated, and blotting the gel onto a nylon or nitrocellulose membrane (preferably nylon). anti-AAV capsid antibodies are then used as primary antibodies that bind to the denatured capsid protein, preferably anti-AAV capsid monoclonal antibodies, most preferably B1 anti-AAV 2 monoclonal antibodies (Wobus et al, J. Virol., 2000)74: 9281-9293). A secondary antibody is then used which binds to the primary antibody and contains a means for detecting binding to the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound thereto, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used for semi-quantitatively determining binding between a primary antibody and a secondary antibody, preferably a detection method capable of detecting radioisotope emission, electromagnetic radiation or colorimetric change, most preferably a chemiluminescent detection kit. For example, for SDS-PAGE, samples can be taken from column fraction fractions and heated in SDS-PAGE loading buffer containing a reducing agent (e.g., DTT) and the capsid proteins resolved on a preformed gradient polyacrylamide gel (e.g., Novex). Silver staining can be made according to manufacture using silver xpress (Invitrogen, California (CA))The description of the quotient proceeds. In one embodiment, the concentration of AAV vector genomes (vg) in the column fraction can be measured by quantitative real-time PCR (Q-PCR). The samples were diluted and digested with DNase I (or another suitable nuclease) to remove the exogenous DNA. After nuclease inactivation, primers and TaqMan specific for the DNA sequence between the primers are used^TMThe fluorescent probe further dilutes and amplifies the sample. The number of cycles (threshold cycles, Ct) required for each sample to reach a defined fluorescence level was measured on an Applied Biosystems Prism 7700 sequence detection system. Plasmid DNA containing sequences identical to those contained in the AAV vector was used to generate a standard curve in the Q-PCR reaction. The value of the cycle threshold (Ct) obtained from the sample is used to determine the vector genome titer by normalizing it against the Ct value of the plasmid standard curve. Digital PCR-based endpoint determination may also be used.

In one aspect, provided herein are optimized q-PCR methods that utilize a broad spectrum serine protease, e.g., proteinase K (e.g., commercially available from Qiagen). More specifically, the optimized qPCR genomic titer assay was similar to the standard assay except that after DNase I digestion, the samples were diluted with proteinase K buffer and treated with proteinase K, followed by heat inactivation. Suitably, the sample is diluted with proteinase K buffer in an amount equal to the size of the sample. Proteinase K buffer can be concentrated 2-fold or more. Typically, proteinase K treatment is about 0.2mg/mL, but can vary from 0.1g/mL to about 1 mg/mL. The treatment step is typically carried out at about 55 ℃ for about 15 minutes, but may be carried out at a lower temperature (e.g., about 37 ℃ to about 50 ℃) for a longer period of time (e.g., about 20 minutes to about 30 minutes), or at a higher temperature (e.g., up to about 60 ℃) for a shorter period of time (e.g., about 5 to 10 minutes). Similarly, heat inactivation is typically at about 95 ℃ for about 15 minutes, but the temperature may be reduced (e.g., about 70 ℃ to about 90 ℃) and the time extended (e.g., about 20 minutes to about 30 minutes). The samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in standard assays.

Additionally or alternatively, droplet digital pcr (ddpcr) may be used. For example, methods have been described for determining single-stranded and self-complementary AAV vector genomic titers by ddPCR. See, e.g., m.lock et al, "methods of human gene therapy" 2014 for 4 months; 25(2) 115-25.doi:10.1089/hgtb.2013.131.Epub 2014.2/14.

5.1.3 production

Hldlr vectors can be made according to the scheme depicted in figure 11. Briefly, cells (HEK 293 cells) were propagated and transfected in a suitable cell culture system to produce vectors. The raav.hldlr vector can then be harvested, concentrated and purified to make a large number of vectors that are then filled and completed in downstream processes.

Methods for making the gene therapy vectors described herein include methods well known in the art, such as generating plasmid DNA for use in generating gene therapy vectors, generating vectors, and purifying vectors. In some embodiments, the gene therapy vector is an AAV vector, and the plasmids produced are an AAV cis plasmid encoding the AAV genome and the gene of interest, an AAV trans plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid. The vector production process may comprise process steps such as starting cell culture, performing cell passaging, seeding cells, transfecting cells with plasmid DNA, exchanging post-transfection medium for serum-free medium, and harvesting the cells and medium containing the vector. The harvested vector-containing cells and culture medium are referred to herein as a crude cell harvest.

Thereafter, the crude cell harvest may be the subject process steps, such as concentration of the carrier harvest, diafiltration of the carrier harvest, microfluidization of the carrier harvest, nuclease digestion of the carrier harvest, filtration of the microfluidized intermediate, purification by chromatography, purification by ultracentrifugation, buffer exchange and formulation by tangential flow filtration and filtration to prepare a large number of carriers.

In certain embodiments, methods similar to those of fig. 11 can be used in conjunction with other AAV producing cells. Many methods are known in the art for producing rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems comprising adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. See, e.g., G Ye et al, clinical development of human Gene therapy (Hu Gene Ther Clin Dev), 25:212-217 (12 months 2014); RM Kotin, Hu Mol Genet,2011, Vol 20, revision Release 1, R2-R6; mietzsch et al, human Gene therapy 25:212-222 (3 months 2014); t Virag et al, human Gene therapy, 20: 807-; clement et al, human Gene therapy, 20:796-806(2009, 8 months); DL Thomas et al, human Gene therapy, 20:861-870 (8 months 2009). rAAV production cultures for production of rAAV viral particles all require: 1) in the case of baculovirus production systems, suitable host cells include, for example, human cell lines such as HeLa, A549 or 293 cells, or insect cell lines such as SF-9; 2) suitable helper virus functions provided by wild-type or mutant adenoviruses (e.g. temperature sensitive adenovirus), herpes viruses, baculoviruses or nucleic acid constructs providing trans or cis helper functions; 3) functional AAV rep genes, functional cap genes and gene products; 4) a transgene (e.g., a therapeutic transgene) flanked by AAV ITR sequences; and 5) suitable media and media compositions for supporting production of rAAV.

Various suitable cells and cell lines have been described for use in AAV production. The cell itself may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Particularly desirable host cells are selected from any mammalian species, including but not limited to cells such as a549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK293 cells (expressing functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). In certain embodiments, the cells are suspension-adapted cells. The choice of the mammalian species from which the cells are provided is not a limitation of the present invention; nor are mammalian cell types, i.e., fibroblasts, hepatocytes, tumor cells, etc.

In specific embodiments, methods for making gene therapy vectors are described in example 3, section 8, below.

5.2 patient population

Patients that are candidates for treatment are preferably adults (male or female aged 18 years ≧ 18) who are diagnosed as having HoFH carrying two LDLR gene mutations; that is, patients with molecularly defined LDLR mutations at both alleles in the context of a clinical manifestation consistent with HoFH, which may include untreated LDL-C levels (e.g., LDL-C levels >300mg/dl), treated LDL-C levels (e.g., LDL-C levels <300mg/dl), and/or total plasma cholesterol levels greater than 500mg/dl, as well as premature and aggressive atherosclerosis. In some embodiments, patients <18 years of age may be treated. In some embodiments, the patient treated is a male aged 18 years or older. In some embodiments, the patient treated is a female aged 18 years or older. Treatment candidates include HoFH patients undergoing treatment with lipid lowering drugs, such as statins, ezetimibe, bile acid sequestrants, PCSK9 inhibitors, and LDL and/or plasma apheresis.

Prior to treatment, HoFH patients should be evaluated for nabs against AAV serotypes for delivery of the hLDLR gene. Such nabs interfere with transduction efficiency and reduce therapeutic efficacy. HoFH patients with baseline serum NAb titers of ≤ 1:10 are good candidates for treatment with rAAV.hLDLR gene therapy regimens. However, in some cases patients with higher ratios may be selected. Treatment of HoFH patients with serum NAb titers >1:5 may require combination therapy, such as temporary co-treatment with immunosuppressive agents, although such therapy may be selected for patients with lower rates. Immunosuppressive agents for such co-therapies include, but are not limited to, steroids, antimetabolites, T cell inhibitors, and alkylating agents. For example, such temporary treatment may comprise administering a steroid (e.g., prednisone) once a day for 7 days in a gradually decreasing dose, starting at about 60mg and decreasing (not administered on day 7) by 10 mg/day. Other dosages and medications may be selected.

The subject may be allowed to continue with its standard of care treatment (e.g., LDL apheresis and/or plasmapheresis, and other lipid lowering treatments) prior to or concurrently with gene therapy treatment, as may be appropriate to their attending physician. In the alternative, the physician may be inclined to stop standard of care treatment prior to administration of the gene therapy treatment, and optionally to resume standard of care treatment as a co-treatment after administration of the gene therapy. The desired endpoints of gene therapy regimens are a reduction in low density lipoprotein cholesterol (LDL-C) and a change in Fractional Catabolism Rate (FCR) of LDL apolipoprotein b (apob) from baseline for up to 12 weeks after administration of gene therapy treatment. Other desirable endpoints include, for example, a reduction in one or more of the following: total Cholesterol (TC), non-high density lipoprotein cholesterol (non-HDL-C), a decrease in fasting Triglycerides (TG) and a change in HDL-C (e.g., increased levels are desired), very low density lipoprotein cholesterol (VLDL-C), lipoprotein (a) (lp (a)), apolipoprotein b (apob), and/or apolipoprotein a-I (apoA-I).

In one embodiment, the patient reaches the desired LDL-C threshold (e.g., LDL-C <200mg/dl, <130mg/dl, or <100mg/dl) after treatment with aav8.hldlr alone and/or in combination with the use of additional treatment for the duration of the study.

In certain embodiments, the patient's need for lipid lowering therapy (including the frequency of LDL and/or plasma apheresis) will be reduced.

In still other embodiments, the number, size, or extent of xanthoma may be assessed to be reduced compared to baseline.

However, at the discretion of the patient care physician, patients with one or more of the following characteristics may be excluded from treatment:

● defined by the NYHA classification as functional grade III and having a hospital history or functional grade IV heart failure within 12 weeks after the baseline visit. .

● medical history within 12 weeks after baseline visit: myocardial Infarction (MI), unstable angina resulting in hospitalization, coronary artery bypass graft surgery (CABG), Percutaneous Coronary Intervention (PCI), uncontrolled arrhythmia, carotid surgery or stent implantation, stroke, transient ischemic attack, carotid revascularization, endovascular surgery or surgical intervention. .

● uncontrolled hypertension is defined as: systolic pressure >180mmHg, diastolic pressure >95 mmHg.

● liver cirrhosis or chronic liver disease history based on recorded histological evaluation or non-invasive imaging or testing.

● documented diagnosis of any of the following liver diseases: non-alcoholic steatohepatitis (biopsy confirmed), alcoholic liver disease, autoimmune hepatitis, liver cancer, primary biliary cirrhosis, primary sclerosing cholangitis, Wilson's disease, hemochromatosis, alpha₁Antitrypsin deficiency.

● abnormal LFT (AST or ALT >2 times the Upper Limit of Normal (ULN) and/or total bilirubin >1.5 times ULN when screened, unless the patient has unbound hyperbilirubinemia due to Gilbert's syndrome.

● hepatitis B by positive definition of HepB SAg, Hep B core Ab and/or viral DNA, or chronic active hepatitis C by positive definition of HCV Ab and viral RNA.

● 52 history of alcohol abuse over a period of weeks.

● certain illicit drugs are known to have potential hepatotoxicity, particularly those that cause steatosis of either microvesicles or macrovesicles. These include, but are not limited to: isotretinoin (acutane), amiodarone, HAART drug, high use of acetaminophen (2 g/day >3 × q weeks), isoniazid, methotrexate, tetracycline, tamoxifen, valproate.

● active tuberculosis, systemic fungal diseases or other chronic infections.

● history of immunodeficiency diseases, comprising positive HIV test results.

● chronic renal insufficiency defined as an estimated GRF <30 ml/min.

● have a history of cancer over the last 5 years, with the exception of well-treated basal cell skin cancer, squamous cell skin cancer or carcinoma of the cervix in situ.

● early organ transplantation.

● any major surgical procedures were performed within 3 months prior to baseline determination and/or treatment.

Baseline serum AAV8 NAb titers >1:5, >1: 10. In other embodiments, the caregiver can determine that the presence of one or more of these physical characteristics (medical history) should not interfere with the treatment provided herein.

5.3. Administration and route of administration

The patient receives a single dose of raav.hldlr administered, e.g., by infusion, e.g., via a peripheral vein, e.g., within about 20 minutes to about 30 minutes. The dose of raav.hldlr administered to the patient was 2.5 × 10¹³GC/kg (as measured by oqPCR or ddPCR).

In certain embodiments, the co-treatment of prophylactic immunomodulation with steroids is initiated at least one day prior to gene therapy (day-1), or on the day of gene therapy delivery (day 0), and continues until about week 8 after administration. In certain embodiments, the prophylactic co-treatment begins at least one day prior to or on the same day as gene therapy delivery and continues at a gradually decreasing dose to about week 13 after administration. Optionally, prophylactic steroid co-therapy may be initiated 2 or 3 days prior to vehicle administration. In certain embodiments, the dose is reduced at a dose of 10 mg/week for each week in weeks 9 and 10, and 5 mg/week for each week in weeks 11, 12 and 13. In certain embodiments, a prophylactic steroid regimen is also delivered when the patient receives a lower dose (e.g., about 2.5 x 1012GC/kg to 7.5 x 1012GC/kg) or a higher dose (e.g., a dose as provided herein). In certain embodiments, another corticosteroid may be substituted for prednisone. In this example, a corticosteroid equivalent dose is provided. For example, a suitable substitute for 40mg of prednisone may comprise betamethasone (about 6mg), cortisone (about 200mg), dexamethasone (about 6mg), hydrocortisone (160mg), methylprednisolone (about 32mg), prednisolone (about 40mg), or triamcinolone acetonide (about 32 mg). Other immunomodulators and dosage equivalents may be determined.

In certain embodiments, administration is initiated one day prior to administration (day-1). The initial dose was 40mg prednisone once a day, gradually decreasing at week 9 and continuing until week 13 is over. The first dose should be administered at least 8 hours prior to the intended administration with the study vehicle on day-1.

Prednisone dose and study week

In certain embodiments, in co-therapy with prednisone or a dose equivalent corticosteroid, the patient receives a regimen comprising at least 2.5 x 10¹²GC/kg or 7.5X 10¹²GC/kg or at least 5X 10¹¹GC/kg to about 7.5X 10¹²Co-therapy of GC/kg (as measured by oqPCR or ddPCR). However, other dosages may be selected.

Optionally, such a regimen may utilize additional immunomodulators. In certain embodiments, such additional immunomodulators are introduced after administration.

In a preferred embodiment, the rAAV suspension used has a potency such that 5 x 10 of a double knockout LDLR-/-Apobec-/-mouse model administered to HoFH (DKO mice)¹¹The GC/kg dose reduced the baseline cholesterol levels in DKO mice by 25% to 75%.

In some embodiments, the dose of raav.hldlr administered to the patient is at 2.5 x 10¹²GC/kg to 7.5X 10¹²In the range of GC/kg. Preferably, the rAAV suspension used has a potency such that it is administered to 5X 10 of a double knockout LDLR-/-Apobec-/-mouse model of HoFH (DKO mice)¹¹The GC/kg dose reduced the baseline cholesterol levels in DKO mice by 25% to 75%. In particular embodiments, the dose of raav.hldlr administered to the patient is at least 5 x 10¹¹GC/kg、2.5×10¹²GC/kg、3.0×10¹²GC/kg、3.5×10¹²GC/kg、4.0×10¹²GC/kg、4.5×10¹²GC/kg、5.0×10¹²GC/kg、5.5×10¹²GC/kg、6.0×10¹²GC/kg、6.5×10¹²GC/kg、7.0×10¹²GC/kg or 7.5X 10¹²GC/kg。

In some embodiments, the raav.hldlr is administered in combination with one or more therapies for treating HoFH. In some embodiments, the raav.hldlr is administered in combination with a standard lipid lowering therapy for the treatment of HoFH, including, but not limited to, statins, ezetimibe, ezedia, bile acid sequestrants, LDL apheresis, plasma apheresis, plasmapheresis, lomitapide, milbexane, and/or PCSK9 inhibitors. In some embodiments, the aldlr is administered in combination with niacin. In some embodiments, the raav.hldlr is administered in combination with a fibrate.

5.4. Measuring clinical goals

The safety of the gene therapy vector after administration can be assessed by the number of adverse events, changes in physical records, and/or clinical laboratory parameters at various time points up to about 52 weeks after vector administration. Although physiological effects may be observed at an early stage (e.g., between about 1 day and one week), in one embodiment, the steady state level of expression is reached at about 12 weeks.

Hldlr administration can be assessed as a defined percentage change in LDL-C over about 12 weeks or at other desired time points, as compared to baseline.

Other lipid parameters compared to baseline values, particularly the percent change in Total Cholesterol (TC), non-high density lipoprotein cholesterol (non-HDL-C), HDL-C, fasting Triglycerides (TG), very low density lipoprotein cholesterol (VLDL-C), lipoprotein (a) (lp (a)), apolipoprotein B (apoB), and apolipoprotein A-I (apoA-I), may also be assessed at about 12 weeks or at other desired time points. The metabolic mechanism by which LDL-C is reduced can be assessed by performing LDL kinetics studies before and after 12 weeks of raav. The main parameter to be assessed is the Fractional Catabolism Rate (FCR) of LDL apoB.

As used herein, the raav.hldlr vectors herein "functionally replace" or "functionally complement" a patient's defective LDLR with active LDLR when the patient expresses sufficient levels of LDLR to achieve at least one of these clinical endpoints. Expression levels of hLDLR that achieve normal wild-type clinical endpoint levels as low as about 10% to less than 100% in non-FH patients may provide a functional alternative.

In one embodiment, expression may be observed as early as about 8 hours to about 24 hours after administration. One or more of the above-described desired clinical effects may be observed within several days to several weeks after administration.

Hldlr administration can be followed by assessment of long-term (up to 260 weeks) safety and efficacy.

Standard clinical laboratory assessments and other clinical assays described in sections 6.4.1 to 6.7 below can be used to monitor adverse events, assess efficacy endpoints for percentage changes in lipid parameters, pharmacodynamic assessments, lipoprotein kinetics, ApoB-100 concentrations, and immune responses to raav.

The following examples are illustrative only and are not intended to limit the present invention.

Examples of the invention

6. Example 1: regimens for treating human subjects

This example relates to gene therapy treatment for patients with gene-confirmed homozygous familial hypercholesterolemia (HoFH) due to Low Density Lipoprotein Receptor (LDLR) gene mutations. . In this example, a gene therapy vector aav8.tbg. hLDLR (a replication defective adeno-associated viral vector 8(AAV8) expressing hLDLR) is administered to a patient with HoFH. Low density lipoprotein cholesterol (LDL-C) levels can be used as a surrogate for transgene expression to assess therapeutic efficacy. The primary efficacy assessment comprised LDL-C levels at about 12 weeks after treatment, and sustained effect for at least 52 weeks thereafter. Long-term safety and persistence of transgene expression can be measured in liver biopsy samples after treatment.

6.1. Gene therapy vector

The gene therapy vector is an AAV8 vector expressing a transgenic human low density lipoprotein receptor (hLDLR) under the control of a liver-specific promoter (thyroxine-binding globulin TBG), and is referred to in this example as aav8.TBG. hLDLR (see fig. 7). The AAV8.TBG. hLDLR vector consists of AAV vector active components and a preparation buffer solution. The external AAV vector component is an icosahedral capsid of serotype 8, T ═ 1, consisting of 60 copies of the three AAV viral proteins VP1, VP2, and VP3 in a ratio of 1:1: 18. The capsid contains a single stranded DNA recombinant aav (raav) vector genome. The genome contains the hLDLR transgene flanked by two AAV inverted terminal repeats (IRTs). Enhancers, promoters, introns, hLDLR coding sequences and polyadenylation (polyA) signals include the hLDLR transgene. ITRs are the genetic elements responsible for replication and packaging of the genome during vector production and are the only viral cis-elements required for rAAV production. Expression of the hLDLR coding sequence was driven from a hepatocyte-specific TBG promoter. Two copies of the α 1 microglobulin/bis kunitz inhibitor enhancer element are located before the TBG promoter to stimulate promoter activity. Chimeric introns were present to further enhance expression, and a rabbit β -globin polyadenylation (polyA) signal was included to mediate termination of hLDLR mRNA transcripts. SEQ ID No. 6 provides the sequence of paav.tbg.pi.hldlrco.rgb used to generate this vector.

The formulations of the test agents were at least 1X 10 in aqueous solution¹³Genome Copies (GC)/mL and administered by infusion via a peripheral vein over 20 minutes (+ -5 minutes) an aqueous solution containing 180mM sodium chloride, 10mM sodium phosphate, 0.001% poloxamer 188, pH 7.3.

6.2. Patient population

The treated patients were adults with homozygous familial hypercholesterolemia (HoFH) carrying two mutations of the LDLR gene. The patient may be a male or female aged 18 years or older. Patients have molecularly defined LDLR mutations at both alleles in the context of clinical manifestations consistent with HoFH, which may include untreated LDL-C levels (e.g., LDL-C levels >300mg/dl), treated LDL-C levels (e.g., LDL-C levels <300mg/dl), and/or plasma total cholesterol levels greater than 500mg/dl, as well as premature and aggressive atherosclerosis. The treated patient may be concurrently treated with a lipid lowering drug, such as a statin, ezetimibe, a bile acid sequestrant, a PCSK9 inhibitor, and LDL apheresis and/or plasma apheresis.

The baseline serum AAV8 neutralizing antibody (NAb) titer of the treated patient can be ≦ 1: 10. If the patient's baseline serum AAV8 neutralizing antibody (NAb) titer is not ≦ 1:10, the patient may be temporarily co-treated with an immunosuppressive agent during the transduction period. In certain embodiments, patients with AAV8 neutralizing antibody titers can be higher (e.g., ≦ 1:5 to ≦ 1:15, or ≦ 1:20) or lower (e.g., ≦ 1:2 to ≦ 1: 5). Immunosuppressive agents for co-therapy include, but are not limited to, steroids, antimetabolites, T cell inhibitors, and alkylating agents.

In still other embodiments, a desired endpoint comprising a reduction in the need for LDL apheresis and/or plasma apheresis is the desired endpoint. The term "LDL apheresis" is used to refer to Low Density Lipoprotein (LDL) apheresis, a process of removing LDL from blood using a process similar to dialysis. LDL apheresis is a procedure that removes LDL cholesterol from a patient's blood. During the LDL apheresis procedure, blood cells are separated from the plasma. A specialized filter is used to remove LDL cholesterol from the plasma and return the filtered blood to the patient. Single LDL apheresis treatment can remove 60% to 70% of harmful LDL cholesterol from the blood. Two machines are currently approved by the food and drug administration in the united states. The fat absorber uses a filter covered with dextran that attaches to and removes LDL from the circulation. Other machines are called HELPs and use heparin to remove LDL. Neither of these machines causes significant changes in the amount of HDL (beneficial) cholesterol. These machinesIs currently approved for patients with a history of coronary artery disease with LDL cholesterol of 2000ng/mdl or higher, and patients without coronary artery disease with LDL cholesterol of 300mg/dl or higher. See, for example, the American Society for Apheresis (American Society for hemodialysis), www.apheresis.com andhttp://c.ymcdn.com/ sites/www.apheresis.org/resource/resmgr/-fact_sheets_file/ldl_apheresis.pdf. See also the World society of plasmapheresis Association (World's plasma dialysis)http:// worldapheresis.org/]And The National Lipid Association (The National Lipid Association) (USA) [ https:// www.lipid.org]. In certain embodiments, plasma apheresis (plasmapheresis) that is non-selective for LDL may have been used prior to gene therapy treatment and the need for such treatment may be reduced, as described herein for LDL apheresis. As used herein, "reduction" of an apheresis refers to a reduction in the number of times per month and/or year that a patient is required to perform an apheresis. Such a reduction may be a 10%, 25%, 50%, 75% or 100% reduction in the apheresis treatment after therapy (e.g., eliminating the need) as compared to the level of apheresis used prior to rAAV8-hLDLR therapy. For example, a patient who has undergone weekly apheresis prior to treatment with raav8.hldlr may only require biweekly, monthly, or less frequent apheresis after treatment. In another example, a patient who has undergone apheresis twice a month prior to treatment with raav8.hldlr may only require monthly, bi-monthly, quarterly, or less frequent apheresis after treatment. And others still

In certain embodiments, a desired endpoint comprises a reduction in the dose of PCSK9 inhibitor used to treat the patient is the desired endpoint. As used herein, "reduction" of an apheresis refers to a reduction in the number of times per month and/or year that a patient is required to perform an apheresis. Such a reduction may be a 10%, 25%, 50%, 75% or 100% reduction in PCSK9 inhibitor required after therapy (e.g., eliminating the need) as compared to the level of PCSK9 inhibitor used prior to rAAV8-hLDLR therapy. For example, treatment of HoFH patients who are infusing a PCSK9 inhibitor monthly (e.g., receiving a 300mg to 500mg dose) prior to raav8.hldlr therapy may result in the ability to reduce treatment with a PCSK9 inhibitor to a therapeutic level consistent with that of HeFH patients. This may enable the patient to receive less invasive therapy (e.g., eliminate the need for high dose infusion). For example, instead of infusing 420 mg/infusion per month, the patient may choose to administer fewer doses (e.g., 100ng/mL to 140ng/mL) using a syringe or auto-injector once a month or once every two weeks (HeFH dose) or with less frequency.

6.3. Administration and route of administration

The patient received a single dose of aav8.tbg. hldlr administered by infusion via the peripheral vein. Aav8.tbg. hldlr is administered to the patient at a dose of about 2.5 × 10¹²GC/kg or 7.5X 10¹²GC/kg. To ensure that empty capsids are removed from the dose of aav8.tbg. hldlr administered to the patient, the empty capsids are separated from the carrier particles by cesium chloride gradient ultracentrifugation or ion exchange chromatography during the carrier purification process as described in section 8.3.2.5.

6.4. Measuring clinical goals

The reduction in LDL-C achieved by administration of aav8.tbg. hldlr can be assessed as a defined percentage change in LDL-C at about 12 weeks compared to baseline.

Other lipid parameters, in particular the percentage of change in Total Cholesterol (TC), non-high density lipoprotein cholesterol (non-HDL-C), HDL-C, fasting Triglycerides (TG), very low density lipoprotein cholesterol (VLDL-C), lipoprotein (a) (lp (a)), apolipoprotein b (apob) and apolipoprotein a-I (apoA-I) can be assessed at about 12 weeks compared to baseline values.

The metabolic mechanisms of LDL-C reduction can be assessed by LDL kinetics studies before vector administration and again at about 12 weeks after administration. The main parameter to be assessed is the Fractional Catabolism Rate (FCR) of LDL apoB.

Long-term (up to 52 weeks or up to 260 weeks) safety and efficacy can be assessed following administration of aav8.tbg. hldlr.

6.4.1. Standard clinical laboratory assessments that can be performed:

the following clinical properties can be tested before and after treatment:

biochemical properties: sodium, potassium, chloride, carbon dioxide, glucose, blood urea nitrogen, Lactate Dehydrogenase (LDH) creatinine, creatinine phosphokinase, calcium, total protein, albumin, aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase, total bilirubin, GGT.

CBC: white Blood Cell (WBC) count, hemoglobin, hematocrit, platelet count, red blood cell distribution width, mean red blood cell volume, mean red blood cell hemoglobin, and mean red blood cell hemoglobin concentration.

Condensation: PT, INR, PTT (at screening and baseline, and as needed).

Urinalysis: urine color, turbidity, pH, glucose, bilirubin, ketones, blood, proteins, WBCs.

6.4.2. Adverse events of interest

The following clinical assays can be used to monitor toxicity:

liver injury

CTCAE v 4.03 grade or higher laboratory results for bilirubin or liver enzymes (AST, ALT, AlkPhos).

Bilirubin and AlkPhos CTCAE v 4.02 grade (bilirubin >1.5 times ULN; AlkPhos >2.5 times ULN).

Hepatotoxicity (i.e., meeting the criteria of "Hai's Law")

AST or ALT ≥ 3 × ULN (upper limit of normal)

>2 × ULN serum Total bilirubin without an increase in alkaline phosphatase

There are no other reasons why elevated transaminase levels and elevated total bilirubin may be explained.

In addition, ALT or AST elevation (>2 fold baseline and 1 fold ULN) that could trigger corticosteroid therapy for putative T cell mediated immune transamination would be labeled and reported.

6.5. End of efficacy

Assessment of the percent change in lipid parameters at about 12 weeks after aav8.tbg. hldlr administration can be assessed and compared to baseline. This includes:

● percent change in LDL-C as directly measured (primary efficacy endpoint).

● Total cholesterol, VLDL-C, HDL-C, calculated percent change in non-HDL-cholesterol, changes in triglycerides, apoA-I, apoB and lp (a).

Baseline LDL-C values can be calculated as the mean of LDL-C levels obtained under fasting conditions in 2 individual cases prior to aav8.tbg. hldlr administration to control laboratory and biological variability and ensure reliable efficacy assessment.

6.5.1. Pharmacodynamic/efficacy assessment

The following efficacy laboratory tests can be evaluated under fasting conditions:

directly measured LDL-C

Lipid group: total cholesterol, LDL-C, non-HDL-C, HDL-C, TG, Lp (a)

Apolipoprotein: apoB and apoA-I.

In addition, optional LDL apoB kinetics can be determined before and 12 weeks after treatment. Lipid lowering efficacy can be assessed as a percentage change from baseline at about 12 weeks, 24 weeks, and 52 weeks after vehicle administration. Baseline LDL-C values were calculated by averaging LDL-C levels obtained under fasting conditions in 2 separate situations prior to administration. The percent change from baseline in LDL-C at 12 weeks after vector administration is the primary measure of gene transfer efficacy.

Change in LDL-apoB fractional catabolism rate from baseline by 12 weeks after administration to vehicle. Additional apoB kinetic parameters will also be considered.

Absolute LDL-C levels at 12 weeks, 24 weeks, 52 weeks and up to 260 weeks per year after administration of aav8. hldlr.

Percent change from baseline in LDL-C and other lipid parameters at 24 weeks, 52 weeks, and up to 260 weeks per year after aav8.hldlr administration.

Percentage of subjects who achieved an absolute LDL-C level <200mg/dl at 12, 24, 52 weeks after aav8.hldlr administration.

The number of subjects who did not recover from a previous dose or did not start any new lipid lowering treatment 12 weeks, 24 weeks, 36 weeks, 52 weeks after aav8.hldlr administration.

The number of subjects undergoing a change in frequency of apheresis treatment at any time during the study for those subjects who underwent lipid apheresis prior to screening.

Comparison of LDL-C achieved after administration of aav8.hldlr to LDL-C achieved when using a PCSK9 inhibitor prior to administration of aav8.hldlr for those subjects receiving a PCSK9 inhibitor.

The amount by which the clinical performance recorded at 12 and 52 weeks after aav8.hldlr administration is improved in number, size or extent for subjects who are easily described for xanthomas at baseline.

6.6. Lipoprotein kinetics

Lipoprotein kinetics studies can be performed before vehicle administration and again after 12 weeks post-administration to assess the metabolic mechanisms of LDL-C lowering. The main parameter to be assessed is the Fractional Catabolism Rate (FCR) of LDL-apoB. Endogenous labeling of apoB was achieved by intravenous infusion of deuterated leucine followed by blood sampling over a 48 hour period.

ApoB-100 isolation

VLDL, IDL and LDL were separated by sequential ultracentrifugation of timed samples drawn after D3-leucine infusion. . Apo B-100 was separated from these lipoproteins by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) using a Tris-glycine buffer system. . The ApoB concentration within an individual ApoB species was determined by enzyme-linked immunosorbent assay (ELISA). The total apoB concentration was determined using automated turbidimetry.

6.6.2 isotopic enrichment determination

The ApoB-100 band was excised from the polyacrylamide gel. The excised band was hydrolyzed in 12N HCl at 100 ℃ for 24 hours. The amino acids were converted to N-isobutyl ester and N-heptafluorobutanamide derivatives prior to analysis using a gas chromatograph/mass spectrometer. Isotopic enrichment (percentage) was calculated from the observed ion flux ratio. Data in this format is similar to the specific radioactivity in a radiotracer experiment. Each subject was assumed to remain in a steady state with respect to apoB-100 metabolism during this procedure.

6.7. Pharmacokinetic and immune response assessment to AAV8

The following tests can be used to assess the pharmacokinetics of AAV vectors, pre-immunization against AAV vectors, and immune responses to AAV vectors:

monitoring of immune response: AAV8 NAb titer; t cell response to AAV8 vector; t cell response to hLDLR.

Concentration of the carrier: AAV8 concentration in plasma measured by PCR in the form of vector genome.

Human leukocyte antigen typing (HLA type): HLA types were assessed from deoxyribonucleic acid (DNA) of Peripheral Blood Mononuclear Cells (PBMC) by high resolution assessment of HLA class I-A, HLA-B, HLA-C and HLA class II DRB1/DRB345, DQB1 and DPB 1. This information allows the potential immune response of T cells to AAV8 capsid or LDLR gene with specific HLA alleles to be correlated, contributing to individual variability in explaining the intensity and timing of T cell responses.

6.8 yellow tumor assessment

Physical examination includes identifying, examining and describing any xanthoma. A record of the location and type of xanthoma, i.e. cutaneous, palpebral (ocular), nodular and/or tendinous, is determined. Metric or calipers are used to record the size (maximum and minimum range) of the xanthoma, where possible, during the physical examination. If possible, a digital photograph of the most widespread and easily identifiable xanthoma could be taken with a tape measure (mm metric) placed near the lesion.

7. Example 2: preclinical data

Non-clinical studies were performed to study the effect of aav8.tbg. hldlr on HoHF and pre-existing animal models of humoral immunity. Multiple single dose pharmacological studies have been performed in small and large animal models measuring cholesterol reduction. Furthermore, regression of atherosclerosis was measured in a double knockout LDLR-/-Apobec 1-/-mouse model (DKO) that lacks both LDLR and Apobec1, even on diet, causes severe hypercholesterolemia due to elevation of LDL containing apob-100, and develops extensive atherosclerosis. These data are used to determine the minimum effective dose and to fully demonstrate dose selectivity in human studies. To further characterize the doses suitable for human studies and identify potential safety signals, toxicology studies were performed in mouse models of non-human primates (NHP) and HoFH.

7.1 preexisting humoral immunity: effect on AAV-mediated Gene transfer to liver

The objective of this study was to assess the effect of pre-existing humoral immunity to AAV on liver-directed gene transfer in rhesus and cynomolgus monkeys. Twenty-one rhesus monkeys and cynomolgus monkeys were selected from a larger population of animals that were pre-screened for levels of preexisting immunity to AAV8. Animals are widely distributed in age and are all male. These studies focused on animals with undetectable low levels of neutralizing antibodies (nabs), while containing a more limited number, with AAV8 NAb titers as high as 1: 160. 3X 10 infusion into animals by peripheral intravenous infusion¹²AAV8 vector at GC/kg expressing Enhanced Green Fluorescent Protein (EGFP) from the liver specific Thyroxin Binding Globulin (TBG) promoter. Animals were necropsied 7 days later and tissues were evaluated for EGFP expression and liver targeting of AAV8 vector genome (figure 1). Pre-existing nabs of AAV8 in NHP sera were assessed using an in vitro transduction inhibition assay and in the context of a passive transfer assay, where sera from NHPs were infused into mice prior to and at the time of vector administration to assess the effect of pre-existing AAV8 nabs on liver-directed gene transfer in vivo (Wang et al, 2010, Molecular Therapy 18(1): 126;. 134).

Animals with pre-existing Nab levels of AAV8 as low as undetectable showed high levels of transduction in the liver as demonstrated by EGFP detection by fluorescence microscopy (figure 1) and ELISA, as well as vector DNA quantification in the liver. In terms of the efficacy of HoFH, the most useful measure of transduction is the percentage of hepatocytes transduced, which in the absence of pre-existing nabs is 17% (range of 4.4% to 40%). This is very close to the efficiency observed in mice with the same dose of vector. Pre-existing Nab that significantly affected hepatocyte transduction had a T threshold titer of < 1:5 (i.e., a titer of 1:10 or greater significantly reduced transduction). Antibody-mediated inhibition of liver transduction is directly associated with a reduction of AAV genomes in the liver. Human sera were screened for evidence of pre-existing NAb to AAV8, and the results showed that approximately 15% of adults had NAb to AAV8 of greater than ≦ 1: 5. Furthermore, it has been demonstrated that higher levels of NAb are associated with changes in the biodistribution of the vector, such that NAb reduces liver gene transfer while increasing vector genome deposition into the spleen without increasing spleen transduction.

7.2AAV8.TBG. mLDLR effects on serum cholesterol in mouse model of HoFH

DKO mice (6 to 12 week old males) were IV injected with aav8.tbg. mldlr and subsequently subjected to metabolic correction and reversal of pre-existing atherosclerotic lesions. Animals were also evaluated for overall clinical toxicity and serum transaminase abnormalities. A mouse version of LDLR was used for vector administration into DKO mice.

Receive 10¹¹GC/mouse (5X 10)¹²GC/kg) showed almost complete return to normal of hypercholesterolemia, which was stable for 180 days (fig. 2). In rodents, no elevation of ALT levels or biochemical abnormalities of the liver were observed up to 6 months after injection of the highest dose of vehicle (Kassim et al, 2010, public science library integrated (PLoS One) 5(10): e 13424).

7.3 effects of aav8.tbg. mldlr on atherosclerotic lesions in mouse models on high fat diet

Considering that AAV 8-mediated LDLR delivery induces a significant reduction in total cholesterol, AAV 8-mediated mLDLR expression was examined in a proof-of-concept study to determine if it had an effect on atherosclerotic lesions (Kassim et al, 2010, public science library, integrated 5(10): e 13424). Three groups of male DKO mice were fed a high fat diet to accelerate the progression of atherosclerosis. Two months later, one group of mice received a single IV injection of 5 × 10¹²GC/kg of control AAV8.TBG. nLacZ vector, one group received a single IV injection of 5X 10¹²GC/kg of aav8.tbg. mldlr vector, while necropsy was performed on the third non-intervention group for atherosclerotic lesion quantification. Mouse quilt with carrierHigh fat diets were maintained for an additional 60 days during which the mice were sacrificed.

Animals receiving aav8.tbg. mldlr vector achieved a rapid decrease in total cholesterol from 1555 ± 343mg/dl at baseline to 266 ± 78mg/dl at day 7 post-treatment and 67 ± 13mg/dl at day 60 post-treatment. In contrast, plasma cholesterol levels remained essentially unchanged in aav8.tbg. nlacz treated mice, from 1566 ± 276mg/dl at baseline to 1527 ± 67mg/dl measured 60 days after vehicle. After two months on a high fat diet, all animals had slightly elevated serum transaminases, which remained elevated after aav8.tbg. nlacz vector treatment, but decreased three-fold to normal levels after aav8.tbg. mldlr vector treatment.

The evolution of pre-existing atherosclerotic lesions was assessed by two independent methods. In the first method, the aorta was opened from the aortic arch to the iliac branch and stained with oil red O (fig. 3A); morphometric analysis quantified the percentage of aorta stained with oil red O along the entire aorta length (fig. 3B). Oil red O is a lysochrome (liposoluble dye) diazo dye used for neutral triglyceride and lipid staining on frozen sections. Staining of the aorta with this dye allows visualization of lipid-loaded plaques. As shown in fig. 3, a two month high fat diet resulted in extensive atherosclerosis covering 20% of the aorta, reflecting baseline disease at vehicle; this increased to 33% over an additional two month period following aav8.tbg. nlacz vector treatment, indicating a further 65% progression of atherosclerosis. In contrast, aav8.tbg. mldlr vector treatment caused atherosclerosis to resolve 87% within two months, with 20% of the aorta covered by atherosclerosis from baseline to only 2.6% of the aorta covered by atherosclerosis 60 days after vector administration.

In the second method, the total lesion area in the aortic root is quantified (fig. 3C-F). This analysis revealed the same general trend, in which mice injected with aav8.tbg. nlacz showed 44% progression within 2 months compared to baseline mice, while mice injected with aav8.tbg. mldlr showed 64% lesion regression compared to baseline mice. Taken together, expression of LDLR by injection of aav8.tbg induced a significant decrease in cholesterol and a significant regression of atherosclerosis at two different sites within the aorta within two months, as assessed by two independent quantitative methods.

7.4HoFH mouse model in the evaluation of the minimum effective dose

Extensive studies of the correlation between phenotype and genotype in the HoFH population have shown that only 25-30% difference in LDL and total cholesterol can translate into significant differences in clinical outcome (Bertolini et al 2013, Atherosclerosis 227(2): 342-. Furthermore, lipid lowering therapy associated with LDL-C reductions of less than 30% can lead to delayed cardiovascular events and prolonged survival in HoFH patients (Raal et al, 2011, cycles 124(20): 2202-2207). Recently, the FDA approved the drug mifepre for treatment of HoFH in which the primary endpoint was a 20% to 25% decrease in LDL-C from baseline (Raal et al, 2010, lancet 375(9719): 998-1006).

In this context, the Minimum Effective Dose (MED) in the gene therapy mouse study discussed below is defined as the lowest dose of vector that results in at least 30% lower serum total cholesterol than baseline, is statistically significant, and is stably reduced. MED has been evaluated in many different studies, and a brief description of each experiment is provided below.

POC dose Range study of AAV8.TBG. mLDLR in DKO mice

Concept dose range validation studies of aav8.tbg.mldlr and aav8.tbg.hldlr in DKO mice were used to determine appropriate doses for further studies. In these studies, DKO male mice were injected intravenously ranging from 1.5 to 500 × 10¹¹GC/kg of various doses of aav8.tbg. mldlr, and subsequent reduction of plasma cholesterol (Kassim et al, 2010, public science library, integrated 5(10): e 13424). These research tests (1.5 to 500X 10)¹¹) The GC dose used in (a) is based on quantitative pcr (qpcr) titers. At day 21, at 1.5X 10¹¹A statistically significant maximum 30% reduction in plasma cholesterol was observed at the GC/kg AAV8 TBG mLDLR doseA large reduction in proportion to larger dose carriers (Kassim et al, 2010, public science library, Integrated 5(10): e 13424). Analysis of liver tissues harvested after metabolic correction revealed that mouse LDLR transgene and protein levels were proportional to vector dose. Thus, a dose-response correlation was observed.

7.4.2DKO and LAHB mice AAV8.TBG. hLDLR dose range Studies

A similar proof of concept study was performed in DKO mice using a vector containing the human LDL receptor (hllr) gene instead of the mouse LDLR receptor gene. The results for the hLDLR vector are very similar to those for mLDLR, i.e., the dose of the vector is proportional to the amount of transgene expression and the amount of vector genome deposited in the liver (Kassim et al, 2013, human Gene therapy 24(1): 19-26). The main difference is in its efficacy-in this model, the efficacy of the human LDLR vector is lower. At 5X 10¹²GC/kg and 5X 10¹¹At GC/kg (dose based on qPCR titers) cholesterol was reduced by at least 30%, although only at higher doses was statistically significant.

The observed decrease in efficacy is attributable to a decrease in the affinity of human LDLR for mouse ApoB. To circumvent this problem, studies repeated the use of LAHB mouse model expressing human ApoB100, thus more realistically mimicking the interaction of human ApoB100 with human LDLR relevant to human studies. Two male mice (DKO vs. lahb) were injected tail vein with one of three vector doses of aav8.tbg. hldlr (0.5 × 10 based on qPCR titer)¹¹GC/kg、1.5×10¹¹GC/kg and 5.0X 10¹¹GC/kg). Animals per syngeneic population were bled on day 0 (before vehicle administration), day 7 and day 21 and serum cholesterol levels were assessed. Compared with an mLDLR DKO mouse, the human LDLR more effective LAHB mouse reduces the serum cholesterol by 30 percent to reach the dosage of 1.5 multiplied by 10¹¹GC/kg, with the same efficacy as the previous studies, achieved DKO animals 2013 with mouse LDLR structure, human Gene therapy 24(1): 19-26).

7.4.3HoFH mouse model of AAV8.TBG.mLDLR and AAV8.TBG.hLDLR non-clinical pharmacology/toxicology research

Male and female DKO mice 6 to 22 weeks old (n 280, 140 male and140 females) received three vector doses (7.5 × 10) of aav8.tbg. mldlr by tail vein injection¹¹GC/kg、7.5×10¹²GC/kg、6.0×10¹³GC/kg) or a dose (6.0X 10) of the intended gene therapy vector AAV8.TBG. hLDLR¹³GC/kg). The administration was done by oqPCR titration based on genomic copy number (GC) per kg body weight, as detailed in section 8.4.1. Additional animals of the same cohort received PBS as a blank. Animals from each group were sacrificed at day 3, 14, 90, 180, respectively, and blood was collected to assess serum cholesterol levels (fig. 4).

A rapid significant decrease in cholesterol was observed at all necropsy time points for all groups of treated mice. At early low dose vectors, this reduction appeared to be less in females than in males, although this difference decreased over time, with no apparent difference between genders. At the same necropsy time point, there was a statistically at least 30% reduction in serum cholesterol for each group compared to the PBS control group. Thus, the MED determined in this study is ≦ 7.5 × 10¹¹GC/kg。

7.4.4 Effect study of homozygous familial hypercholesterolemia mouse model of AAV8.TBG.hLDLR

Four doses (1.5 × 10) of aav8.tbg. hldlr were administered by intravenous infusion in 12 to 16 week old male DKO mice (n ═ 40)¹¹GC/kg、5.0×10¹¹GC/kg、1.5×10¹²GC/kg、5.0×10¹²GC/kg) (dose based on oqPCR titration). Animals were bled and serum cholesterol assessed on day 0 (before vehicle administration), day 7 and day 30 (figure 5). On days 7 and 30, at usage approximately equal to 5.0X 10¹¹A rapid and dramatic reduction in cholesterol was observed in the GC/kg treated mouse cohort. MED values determined according to this study were 1.5X 10¹¹GC/kg and 5.0X 10¹¹Between GC/kg.

7.5. Effect of high fat diet AAV8 TBG rhLDLR in LDLR +/-rhesus monkey

A study designed to evaluate AAV8-LDLR gene transfer in FH rhesus monkeys was performed. Subsequent 10 fat-or feed-fed wild-type rhesus monkey¹³GC/kg of AAV8.TBAdministration of rhAFP (control vehicle; dose based on qPCR titration) did not see elevated aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) values. This suggests that the AAV8 capsid itself is responsible for the process of causing validation or liver injury.

7.6. study of AAV8.TBG. hLDL test-point tissue distribution in HoFH mouse model

To evaluate safety and pharmacodynamic performance for HoFH gene therapy, DKO mice were subjected to a pilot tissue distribution (BD) study. These studies examined systemic administration of 5X 10 by one of two routes¹²Vector distribution and persistence of five female DKO mice of GC/kg aav8.tbg. hldl vector: 1) IV injection into the tail vein or 2) portal vein injection. At two different time points (day 3 and day 28), a set of tissues was harvested and total cellular DNA was extracted from the harvested tissues. In these pilot studies, both the IV and portal routes form comparable BD contour maps, supporting the basic theory of injection of gene therapy vectors into patients and animals via peripheral veins.

7.7. Toxicology

To assess the potential toxicity of gene therapy to HoFH, we performed pharmacological/toxicology studies in DKO mice (HoFH mouse model), wild type, and LDLR +/-rhesus monkeys. These studies include the detection of the role of LDLR transgene expression in vector-associated toxicity in feed wild-type and LDLR +/-rhesus monkeys, pharmacological/toxicology studies of aav8.tbg.mldlr and aav8.tbg.hldlr in HoFH mouse models, and examination of the non-clinical tissue distribution of aav8.tgb.hldlr in HoFH mouse models. These studies are described in more detail below.

7.8. Non-clinical study to examine LDLR transgene expression in vector-associated toxicity of feed-fed wild-type LDLR +/-rhesus monkeys

Four wild-type and four LDLR +/-rhesus monkey intravenous injections 1.25X 10¹³GC/kg aav8.tbg. hldlr (dose based on oqPCR titration), non-human primates (NHP) were monitored for up to one year after administration. Four animals (two wild-type and two LDLR +/-) were necropsied at day 28 post-vector administration to assess acute vector-associated toxicity and vector distribution and four animals (two)Wild type and two LDLR +/-) were necropsied at 364/365 days post vector administration to assess long-term vector-associated pathology and vector distribution. Each cohort of wild type and LDLR +/-macaques had two males and two females.

The animal has good tolerance to the injection carrier and has no long-term or short-term clinical sequelae. Tissue distribution studies indicate high levels and stable liver targeting with less extrahepatic distribution, but still detectable, decreasing over time. These data suggest that the liver, the target organ for drug efficacy, is also the most likely organ to produce potential toxicity. A detailed review of the tissues harvested in necropsies performed at day 28 and day 364/365 after vector administration revealed minimal to mild liver manifestations and some evidence of atherosclerosis in LDLR +/-macaques. The nature of the liver pathologies and the fact that similar pathologies were observed in two untreated wild-type animals indicate to the pathologist that they were not related to the test article.

One animal was continuously elevated in glutamate pyruvate transaminase (ALT) prior to vector administration, and continued to be elevated after vector administration at levels ranging between 58 and 169U/L. The transaminase levels in the remaining animals were not elevated, or aspartate transaminase (AST) and alanine transaminase (ALT) were only briefly elevated at low levels, never exceeding 103U/L. The most consistent abnormalities were found after vector injection, indicating that they are associated with the test article. Activation of human LDLR or AAV8 capsid by T cells correlates with increased AST/ALT. Figure 6 presents AAV capsid ELISPOT data and serum AST levels for three animals demonstrating relevant findings. Only one animal showed a correlation, with an AST increase of 103U/L corresponding to the appearance of T cells against the capsid (FIG. 6, animal 090-0263); the capsid T cell response persists and AST immediately reverts to the normal range.

Analysis of tissue-derived T cells presenting capsid and transgene-specific T cells analysis indicated that liver-derived T cells became responsive to T cells from both genotypes (wild type and LDLR +/-) at a later time point, while human LDLR T cells were detected in LDLR +/-animals at this later time point. This indicates that PBMCs do not reflect T cell compartments in the target tissue. Liver tissues harvested at day 28 and day 364/365 were analyzed for transgene expression by RT-PCR and were found to be indeed affected by clinical pathological abnormalities or T cell appearance.

Neither wild type nor LDLR +/-type animals developed hypercholesterolemia on the diet. No dose limiting toxicity (dlt) was observed at 1.25X 10¹³GC/kg (based on oqPCR), which means that the Maximum Tolerated Dose (MTD) will be equal to or greater than this dose. An increase in test article-associated transaminase was observed, which was low and transient, but still present. Therefore, the No Observed Adverse Effect Level (NOAEL) was less than the single high dose evaluated in this example 1.

Non-clinical pharmacology/toxicology studies of aav8.tbg.mldlr and aav8.tbg.hldlr in HoFH mouse model

This study, performed in DKO mice with this strain, would allow 1) the assessment of proof-of-concept efficacy and toxicity, and 2) the assessment of vector-associated toxicity in any pathological situation associated with LDLR deficiency and associated dyslipidemia and its sequelae, such as steatosis.

This study was designed to test aav8.tbg. hldlr at the highest dose, which is 8 times the highest dose administered to human subjects with HoFH, as described in example 1. The vector version expressing mouse LDLR was tested at this high dose and two low doses to assess the effect of dose on toxicity parameters, as well as the reduction of cholesterol. Dose response experiments were performed using vectors expressing mouse LDLR to more closely reflect the toxicity and efficacy observed in humans using human LDLR vectors.

In this study, male and female DKO mice 6-22 weeks old were administered aav8.tbg. mldlr (7.5 × 10), respectively¹¹Gc/kg、7.5×10¹²Gc/kg and 6.0X 10¹³Gc/kg) or 6.0X 10¹³Vector for GC/kg (aav8.tbg. hldlr) (dose based on oqPCR titration). Animals were sacrificed at day 3, 14, 90 and 180 after vector administration; these times were chosen to capture the test article of the vector expression profile and acute and chronic toxicity. The effectiveness of transgene expression was monitored by measuring serum cholesterol levels. The animal is subjected to comprehensive clinicPathology, immune response to vector (cytokines, NAb of AAV8 capsid, T cell response to capsid and transgene) evaluation, and tissues were harvested at necropsy for comprehensive histopathological examination.

The main toxicological results of this study are as follows:

no clinical sequelae were observed in any of the treatment groups

Clinical pathology

Abnormalities in transaminases were limited to elevations in liver function tests AST and ALT (ranging from 1-4 times ULN) and were found mainly on day 90 of all doses of murine LDLR vector. Transaminases were not elevated in cohorts administered with high doses of human LDLR vector except in a few males where ALT was less than 2 times ULN. The abnormalities associated with the mouse vector were mild, not dose dependent, and therefore not considered vector-related. Essentially no results were found in association with the high dose human vehicle. Based on these findings, there is no evidence of treatment-related toxicity, which means that the no adverse effect level (NOAEL) based on these criteria is 6.0 × 10¹³GC/kg。

Pathology: no obvious pathological results were observed. Only the following minor or mild manifestations were seen in liver histopathological examination:

animals administered PBS had evidence of mild and/or mild abnormalities according to all evaluation criteria. In assessing treatment-related pathology, any findings found in PBS-injected animals classified as mild were of interest.

Mild bile duct hyperplasia and sinus cell hyperplasia were observed in high dose female mice administered with mouse vector and human LDLR vector. This may represent a vehicle-related effect that is only observed at high doses.

Central leaflet hypertrophy, which is considered to be carrier-independent, is mild, is only present in males and not under high dose carriers.

On day 180, males of 1/7 and females of 3/7 found minimal necrosis in the high dose human LDLR vector.

Based on the finding of mild biliary and sinus hyperplasia in high dose vectors, and in high dose human LDLR vectorsExamples of small amounts of necrosis, based on these criteria, NOAEL at 7.5X 10¹²Gc/kg and 6.0X 10¹³Between GC/kg.

Other findings: based on the response of IFN- γ ELISPOT to capsid and LDLR, animals developed evidence of increased NAb of AAV8 and very low T cell response following administration of high doses of human LDLR vector. There was little evidence of an acute inflammatory response based on serum analysis at day 3 and 14 post-vehicle; although IL6 was not increased, some cytokines did show moderate and transient elevations.

One notable finding is that DKO mice treated with the mouse LDLR vector are not more toxic than mice treated with the human LDLR vector, which may be the case if human LDLR is more immunogenic than the mouse transgene in terms of T cells. ELISPOT studies did show some activation of LDLR-specific T cells when using high dose vectors expressing human transgenes in mice, although the T cells were low in number and present in a limited number of animals supporting toxicity data, suggesting that this mechanism of host response is unlikely to lead to safety issues.

In summary, there is no dose limiting toxicity, which means that the maximum tolerated dose is 6.0 × 10 above the highest dose tested¹³GC/kg. Based on mild and reversible findings in liver pathology at the highest dose, NOAEL was 6.0 x 10¹³Between Gc/kg, a slightly reversible pathology was observed in the liver, which dropped to 7.5X 10¹²GC/kg, no clear vector-related findings.

7.10. AAV8.TGB. hLDLR non-clinical tissue distribution of HoFH mouse model

Intravenous administration of 7.5X 10 to 6-week old male and female DKO mice¹²GC/kg of aav8.tbg. hldlr (dose measured by oqPCR titration) (highest dose for treating human subjects in example 1 f). At day 3, 14, 90 and 180 after administration of the vehicle, animals were necropsied for tissue distribution assessment. In addition to blood, 20 organs were harvested. The distribution of the vector genome in the organs was assessed by quantitative, sensitive PCR analysis of the total genomic DNA harvested. To evaluate the PCR testOne sample per tissue was tested for sufficiency of the reaction and included the peak potential of the control DNA, which included a known number of vector sequences.

The number of vector GCs in the liver was significantly higher than other organs/tissues, consistent with the high hepatic nature of AAV8 capsid. For example, at day 90, the vector genome copies in the liver were at least 100-fold that of any other tissue. At the first three time points, the male and female mice differences were not statistically significant. The GC value in the liver decreased with the passage of time until day 90 and then became stable. All tissues showed similar decreases, but the vector copy number decreased more rapidly in tissues with higher cell turnover rates. Low but detectable levels of copies of the vector genome are present in the gonads and brain.

Tissue distribution of aav8.tbg in DKO mice is consistent with published AAV8 results. The liver is the main target for gene transfer after intravenous infusion, and genomic copies in the liver do not significantly decline over time. Other organs were targeted for vector delivery, although the level of gene transfer in these non-liver tissues decreased significantly and over time. Thus, the data presented here indicate that the primary organ system assessment is the liver.

7.11. Conclusions from non-clinical safety studies

Rhesus monkey and DKO mouse studies demonstrated that high doses of vehicle were associated with low-level, transient, asymptomatic liver pathology, manifested as a transient elevation of transaminases in NHPs, and mild biliary and sinus hypertrophy in mice. No other toxicity due to the vector was observed.

In macaques the dosage is up to 1.25X 10¹³At 6X 10 under GC/kg and in DKO mice¹³No DLT was observed at GC/kg. NOAEL was determined to focus mainly on hepatotoxicity as reflected by elevated transaminases in cynomolgus monkeys and histopathology in DKO mice. That is, in macaques the NOAEL is less than 1.25X 10¹³GC/kg and NOAEL less than 6X 10 in DKO mice¹³GC/kg but more than 7.5X 10¹²GC/kg. The dose was based on oqPCR titration.

7.12. Overall evaluation of non-clinical data supporting human therapy

Key findings derived from pharmacological and toxicological studies provide information for dose selection and design for clinical studies, as follows:

minimum Effective Dose (MED): MED, defined as the GC/kg dose in non-clinical studies, reduces serum cholesterol by 30%. Two IND-enabled non-clinical studies were identified 1.5 to 5.0X 10¹¹MED between GC/kg. Pharmacological/toxicological studies in mice relative to PBS control showed statistically significant reductions in serum cholesterol of at least 30%, allowing an MED ≦ 7.5X 10¹¹Estimation of GC/kg. From the observed dose-response relationship, MED values in the range of 1.5 to 5.0X 10 can be determined by oqPCR¹¹Between GC/kg.

Maximum Tolerated Dose (MTD): MTD is defined in non-clinical studies as the GC/kg dose, which does not result in dose-limiting toxicity (DLT). No DLT was observed in toxicology studies at the highest dose tested, as determined by oqPCR, of 6.0 x 10 in DKO mice¹³GC/kg and 1.25X 10 in macaque¹³GC/kg. The results indicate that the actual MTD is higher than these doses.

Mice were given 6.0X 10¹³No adverse effects were seen with the GC/kg dose of aav8.tbg. hldlr, following treatment on day 3, 14, 90 or 180. In monkeys and mice using aav8.tbg. hldlr, there was an occasional increase in transaminases in monkeys and mice. In mice, mild hepatic necrosis was observed in aav8.tbg. hldlr-treated mice only at day 180. However, it was not observed on day 90 or in any animals using the murine transgene product, which may indicate that it may be associated with an immune response to the human transgene product. Whereas no significant adverse reactions were observed in mice and monkeys using aav8.tbg. hldlr, minor elevations in ALT and AST were consistent with clinical data describing the potential for AAV to trigger liver effects.

No adverse event rating (NOAEL) observed: this was determined to be 7.5 × 10 in DKO mice¹²GC/kg. This is based on the fact that it is small to small, mainly in the liver (bile ducts and sinus hyperplasia, micro necrosis)Mild histopathological findings, high doses of human ldlr (hldlr) transgene were observed. Only one dose was tested in macaques; however, 1.25X 10¹³Toxicity under GC/kg was minimal, containing a temporary and low level elevation of AST and ALT, indicating that true NOAEL would be achieved at doses lower than the tested dose.

Based on these data, three single dose cohorts, 2.5 × 10, were proposed¹²GC/kg、7.5×10¹²GC/kg and 2.5X 10¹³GC/kg (dose based on oqPCR method). These doses represent the semilog of dose responses that can be developed, escalated, and represent dose ranges not supported by clinical testing. The introduction of prophylactic glucocorticoids in clinical protocols is expected to improve the safety of product administration by reducing or preventing immune-mediated liver cell damage.

8. Example 3: manufacture of aav8.tbg. hldlr

The AAV8.TBG. hLDLR vector consists of AAV vector active components and a preparation buffer solution. The external AAV vector component is an icosahedral capsid of serotype 8, T ═ 1, consisting of 60 copies of the three AAV viral proteins VP1, VP2, and VP3 in a ratio of 1:1: 18. The capsid contains a single stranded DNA recombinant aav (raav) vector genome (fig. 7). The genome contains a human Low Density Lipoprotein Receptor (LDLR) transgene flanked by two AAV Inverted Terminal Repeats (ITRs). Enhancers, promoters, introns, human LDLR coding sequence and polyadenylation (polyA) signals include the human LDLR transgene. ITRs are genetic elements responsible for replication and packaging of the genome during vector production and are the only viral cis-elements required for rAAV production. Expression of the human LDLR coding sequence was driven from a hepatocyte-specific thyroxin-binding globulin (TBG) promoter. Two copies of the α 1 microglobulin/bis kunitz inhibitor enhancer element are located before the TBG promoter to stimulate promoter activity. Chimeric introns were present to further enhance expression, and a rabbit β -globin polyA signal was included to mediate termination of human LDLR mRNA transcripts. The vector was supplied as a suspension of aav8.tbg. hldlr vector in the formulation buffer. The buffer solution was prepared with 180mM sodium chloride, 10mM sodium phosphate, 0.001% poloxamer 188, and pH 7.3.

Details of the carrier fabrication and carrier characterization are described in the following sections.

8.1. Plasmid for producing AAV8.TBG.hLDLR

The plasmids used to generate aav8.tbg. hldlr were as follows:

8.1.1 cis plasmid (vector genome expression construct):

penn.aav.tbg.hldlr.rbg.kanr containing the human LDLR expression cassette (fig. 8). This plasmid encodes a rAAV vector genome. The polyA signal used for the expression cassette was from the rabbit β globin gene. Two copies of the α 1 microglobulin/bis kunitz inhibitor enhancer element precede the TBG promoter.

To generate the cis plasmid used to generate aav8.tbg.hldlr, the human LDLR cDNA was cloned into the construct penn.aav.tbg.pi containing AAV2 ITR to create penn.aav.tbg.hldlr.rbg. plasmid backbone of penn.aav.tbg.pi was derived from pzac2.1 (pKSS based plasmid). The ampicillin resistance gene in penn.aav.tbg.hldlr.rbg was excised and replaced with the kanamycin gene to create penn.aav.tbg.hldlr.rbg.kanr. Expression of the human LDLR cDNA was driven by a TBG promoter with a chimeric intron (Promega Corporation, wisconsin, usa). The polyA signal used for the expression cassette was from the rabbit β globin gene. Two copies of the α 1 microglobulin/bis kunitz inhibitor enhancer element precede the TBG promoter.

Description of sequence elements

1. Inverted Terminal Repeat (ITR): AAV ITRs (GenBank # NC001401) are sequences identical at both ends but oriented in opposite directions. When AAV and adenovirus (ad) helper functions are provided in trans, the AAV2 ITR sequences serve as the origin of vector DNA replication and packaging signals for the vector genome. Thus, the ITR sequences represent the only cis-acting sequences required for vector genome replication and packaging.

2. The human alpha 1 microglobulin/bichonitz inhibitor enhancer (2 copies; 0.1 Kb; GenBank # X67082), a liver-specific enhancer element, contributes to the enhancement of liver specificity and the enhancement of expression from the TBG promoter.

3. The human thyroxine-binding globulin (TBG) promoter (0.46 Kb; GenBank # L13470). This hepatocyte-specific promoter drives expression of the human LDLR coding sequence.

4. Human LDLR cDNA (2.58 Kb; GenBank # NM000527, complete CDS). The human LDLR cDNA encodes a low density lipoprotein receptor of 860 amino acids, with an SDS-PAGE predicted molecular weight of 95kD and an apparent molecular weight of 130 kD.

5. Chimeric introns (0.13 Kb; GenBank # U47121; Promega, Wisconsin, Madison). The chimeric intron consists of a5 '-donor site from the first intron of the human β -globin gene, and a branch of the intron and a 3' -acceptor site located between the leader and the body of the heavy chain variable region of the immunoglobulin gene. The presence of introns in the expression cassette has been shown to facilitate transport of mRNA from the nucleus to the cytoplasm, thereby increasing the accumulation of stable levels of mRNA for translation. This is a common feature of gene vectors that aim to regulate the increase in gene expression levels.

6. Rabbit β -globulin polyadenylation signal: (0.13 Kb; GenBank # V00882.1) Rabbit beta-globulin polyadenylation signal provides a cis sequence for efficient polyadenylation of antibody mRNA. This element serves as a signal for transcription termination, a specific cleavage event at the 3' end of the nascent transcript, and a signal for the addition of a long poly A tail.

8.1.2 Trans plasmid (packaging construct): pAAV2/8(Kan), containing AAV2 rep gene and AAV8 cap gene (FIG. 9).

AAV8 trans plasmid pAAV2/8(Kan) expresses AAV2 replicase (rep) genes and AAV8 capsid (cap) genes encoding virion proteins VP1, VP2 and VP 3. AAV8 capsid gene sequences were originally isolated from rhesus monkey heart DNA (genbank accession No. AF 513852). To create the chimeric packaging construct, plasmid p5E18 containing AAV2 rep and cap genes was digested with XbaI and XhoI to remove AAV2 cap genes. The AAV2 capsid gene was then replaced with a 2.27Kb SpeI/XhoI PCR fragment of the AAV8 cap gene to create plasmid p5E18VD2/8 (FIG. 9 a). The AAV p5 promoter, which typically drives rep expression, migrates in this construct from the 5 'end of the rep gene to the 3' end of the cap gene. This arrangement is to down-regulate expression of rep to improve vector yield. The plasmid backbone for p5E18 was from pBluescript KS. As a final step, the ampicillin resistance gene was replaced with the kanamycin resistance gene to create pAAV2/8(Kan) (fig. 9B). The entire pAAV2/8(Kan) trans plasmid was verified by direct sequencing.

8.1.3 adenovirus helper plasmids: pAd. DELTA.F 6(Kan)

Plasmid pAd Δ F6(Kan) was 15.7Kb in size and contained regions of the adenovirus genome important for AAV replication, i.e., E2A, E4, and VA RNA. pAd. DELTA.F 6(Kan) does not encode any additional adenovirus replication or structural genes and does not contain cis-elements necessary for replication, such as adenovirus ITRs, and therefore, infectious adenovirus is not expected to be produced. The functions of the essential genes of adenovirus E1 are provided by HEK293 cells producing rAAV vectors. pAd Δ F6(Kan) was derived from E1, E3 deleted molecular clone of Ad5 (pBHG10, pBR322 based plasmid). Deletions were introduced into Ad5 DNA to remove unnecessary adenoviral coding regions and reduce the amount of adenoviral DNA in the helper plasmid from 32Kb to 12 Kb. Finally, the ampicillin resistance gene was replaced with the kanamycin resistance gene to create pAd Δ F6(Kan) (fig. 10). DNA plasmid Sequencing was performed by Qiagen Sequencing Services, Germany, and revealed a 100% homology of the pAdDeltaF6(Kan) reference sequence to the following adenoviral elements: p1707FH-Q E4 ORF 63.69-2.81 Kb; E2A DNA binding protein 11.8-10.2 Kb; VA RNA region 12.4-13.4 Kb.

Each of the cis, trans and helper plasmids described above contains a kanamycin resistance cassette, and therefore the β -lactam antibiotic is not used for its production.

8.1.4 plasmid production

All plasmids used to generate vectors were produced by pureyn Inc (pureyn Inc.) (Malvern, PA, usa). All growth media used in the process were animal independent. All components used in the process, including the fermentation flask, vessel, membrane, resin, column, tube, and any components in contact with the plasmid, were specific for a single plasmid and were certified BSE-free. There are no shared components and disposable components are used when appropriate.

8.2. Cell bank

Aav8.tbg. hldlr vectors were generated from HEK293 working cell bank derived from a well characterized master cell bank. Details of the manufacture and testing of the two cell banks are shown below.

8.2.1HEK293 Master cell Bank

HEK293 Master Cell Bank (MCB) is a derivative of primary human embryonic kidney cells (HEK) 293. The HEK293 cell line is a permanent line which has been DNA-splicing transformed by human adenovirus type 5 (Ad5) (Graham et al, 1977, Journal of General Virology 36(1): 59-72). HEK293 MCB has been commonly tested for microbial and viral contamination. HEK293 MCB is currently stored in liquid nitrogen. Additional tests were performed on HEK293 MCB to demonstrate the absence of specific pathogens of human, simian, bovine and porcine origin. Human origin of HEK293 MCB was confirmed by isozyme analysis.

HEK293 MCB was also tested for tumorigenicity by assessing tumor formation in nude (nu/nu) thymus mice after subcutaneous injection of cell suspensions. In this study, ten of ten positive control mice diagnosed fibrosarcoma at the injection site, and ten of ten test mice diagnosed cancer at the injection site. None of the mice in the negative control group were diagnosed with tumors. HEK293 MCB L/N3006-105679 was also tested for the presence of Porcine Circovirus (PCV) type 1 and type 2. Both PCV type 1 and type 2 MCB were negative.

8.2.2HEK293 working cell Bank

The HEK293 Working Cell Bank (WCB) was manufactured using new zealand-derived fetal bovine serum FBS (Hyclone PN sh30406.02) certified for applicability to the european pharmacopeia monograph. One vial (1mL) of MCB was used to establish HEK293 WCB for the seeding material. Characterization tests were performed and the test results are listed

Table 4.1.

Table 4.1 characterization of HEK293 WCB.

8.3. Carrier manufacture

A general description of the carrier manufacturing process is given below and is also reflected in the flow chart of fig. 11.

8.3.1 vector production Process (upstream Process)

8.3.1.1 HEK293 WCB cells were initially cultured in T flasks (75 cm)²) In

One vial of 10 from WCB⁷Individual 1mL HEK293 cells were thawed at 37 ℃ and plated to 75cm²The tissue culture flask of (a), the flask containing DMEM high glucose (DMEM HG/10% FBS) supplemented with 10% fetal bovine serum. Cells were then placed at 37 ℃/5% CO₂Incubators, and grown to about 70% confluence, direct observation and microscopy daily to assess cell growth. These cells were designated for passage 1 and passaged to produce a cell seeding train for vector biosynthesis for up to about 10 weeks, as described below. The number of passages was recorded at each passage and cells were terminated after the 20 th passage. If additional cells are required for vector biosynthesis, a new seeding train of HEK293 cells is initiated from another vial of HEK293 WCB.

8.3.1.2 cells were passaged to-2T flasks (225 cm)²) In

When HEK293 cells were grown to 70% confluence in T75 flasks, the cells were detached from the surface of the flasks using recombinant trypsin (TrypLE) and seeded into two T225 flasks containing DMEM HG/10% FBS. Cells were placed in an incubator and incubated to 70% confluence. The growth, presence or absence of contamination and uniformity of the cells were observed by naked eye observation and using a microscope.

8.3.1.3 cells were passaged to-10T flasks (225 cm)²) In

When HEK293 cells reach 70% confluence in two T225 flasks, cells were detached using recombinant trypsin (TrypLE) and plated at-3X 10 per flask⁶The density of each cell was 225cm with ten DMEM HG/10% FBS²And sowing seeds in the T flask. Place the cells in 37 ℃/5% CO₂Incubators were incubated to 70% confluency. The growth, absence of contamination and consistency of the cells were monitored by direct visual inspection and using a microscope. Cells are passed throughSerial passages in T225 flasks maintained cell seed culture and provided cells for expansion to support the manufacture of subsequent vector batches.

8.3.1.4 cells were passaged into-10 roller bottles

When 70% of HEK293 cells were aggregated in ten T225 flasks, the cells were detached with recombinant trypsin (TrypLE), counted and seeded at 850cm containing DMEM HG/10% FBS²Roller Bottle (RB). RB was then placed in the RB incubator and cells were grown to-70% confluency. Cell growth, absence of contamination and consistency were monitored by direct visual inspection and using a microscope.

8.3.1.5 cells were passaged into-100 roller bottles

When HEK293 cells were grown in RB prepared in the above procedure to a concentration of-70%, they were separated using recombinant trypsin (TrypLE), counted and seeded in 100 RBs containing DMEM/10% FBS. RB was then placed in an RB incubator (37 ℃, 5% CO)₂) Culturing until the confluence reaches 70%. The growth, absence of contamination and consistency of the cells were monitored by direct visual inspection and using a microscope.

8.3.1.6 transfection of cells with plasmid DNA

When HEK293 cells grown in 100RB were-70% confluent, the cells were transfected with three plasmids, an AAV serotype specific packaging (trans) plasmid, an ad helper plasmid, a vector cis plasmid containing the human LDLR gene expression cassette flanked by AAV Inverted Terminal Repeats (ITRs). Transfection was performed using the calcium phosphate method (for plasmid details, see section 4.1.1). RB was placed in an RB incubator (37 ℃, 5% CO)₂) Overnight.

8.3.1.7. Medium exchange to serum-free medium

After overnight incubation of 100 RBs after transfection, the medium containing DMEM/10% FBS with transfection reagent was removed from each RB by aspiration and replaced with DMEM-HG (without FBS). RB was returned to the RB incubator and 5% CO at 37 ℃₂Incubate until harvest.

8.3.1.8. Vector harvest

RB were removed from the incubator and examined for evidence of transfection (transfection-induced cell morphology change, cell monolayer detachment) and for evidence of any contamination. Cells were detached from the RB surface by stirring each RB and then harvested by slowly pouring into a disposable sterile funnel connected to a bioprocess container (BPC). The combined harvest material in BPC was labeled as' product intermediate: crude cell harvest' and samples were collected for (1) in-process bioburden testing and (2) bioburden, mycoplasma and adventitious factor product release testing. The product intermediate batch labeled as crude Cell Harvest (CH) was stored at 2 ℃ to 8 ℃ until further processing.

8.3.2 purification of the vector (downstream Process)

While a common "platform" purification process is used for all AAV serotypes (i.e., pooling the same series and step sequence), each serotype requires unique chromatography step conditions, which also affects some details of the steps (buffer composition and pH) used to prepare a clarified cell lysate for the chromatography resin.

8.3.2.1AAV8 vector harvest concentration and diafiltration by TFF

BPC with crude CH was connected to the inlet of the sterile reservoir of a hollow fiber (100k MW cutoff) TFF unit, which was in equilibrium with phosphate buffered saline. The crude CH was applied to the TFF apparatus by peristaltic pump and concentrated to 1-2L. The support is retained (retentate), while the small molecular weight fraction and the buffer substance pass through the pores of the TFF filter and are discarded. The harvest was then diafiltered with AAV8 filter buffer. After diafiltration, the concentrated carrier was recovered in 5L BPC. The material was labeled as' product intermediate: after TFF, harvest' and samples were collected for in-process bioburden testing. The concentrated harvest is immediately further processed or stored at 2-8C until further processing.

8.3.2.2 microfluidization and nuclease digestion of the harvest

The concentrated and diafiltered harvest was subjected to shear using a microfluidizer, which broke whole HEK293 cells. The microfluidizer is sterilized with 1N NaOH for at least 1 hour after each use, stored in 20% ethanol until the next run, and rinsed with WFI before each use. Coarse fraction contained in BPCThe carrier is attached to the sterile inlet of the microfluidizer and sterile air BPC is attached to the outlet port. Cells containing the carrier were passed through a microfluidizer interaction chamber (a cyclotron channel 300 μm in diameter) to lyse the cells and release the carrier using air pressure generated by the microfluidizer. The microfluidization process is repeated to ensure complete lysis of the cells and high recovery of the carrier. The product intermediate was repeatedly passed through a microfluidizer and washed with-500 mL AAV8 benzene synthase buffer. The 5L BPC containing microfluidization carrier from the microfluidization device outlet separation. This material was labeled as' product intermediate: final microfluidization' and sample collection for in-process bioburden testing. The microfluidized product intermediate is further processed immediately or stored at 2 ℃ to 8 ℃ until further processing. By adding 100U/mLNucleic acid impurities were removed from AAV8 particles. The contents of the BPC were mixed and incubated at room temperature for at least 1 hour. And (4) further processing the enzymatic hydrolysis product intermediate.

8.3.2.3 filtration of microfluidized intermediates

BPC containing microfluidization and digestion product intermediates was attached to a cartridge filter with a pore size gradient from 3 μm to 0.45 μm. Filters were conditioned with AAV benzoylase buffer. Using a peristaltic pump, the microfluidized product intermediate is passed through a cartridge filter and collected in a BPC connected to the filter outlet. Sterile AAV8 benzoylase buffer was passed through the cartridge to flush the filter. The filtered product was then intermediately ligated into a 0.2 μm final pore size capsule filter conditioned with AAV 8-benzzyme buffer. The filtered intermediate was passed through a cartridge filter using a peristaltic pump and collected in a BPC connected to the filter outlet. A large amount of sterile AAV8 benzoylase buffer was pumped into the filter element to flush the filter. The material was labeled as' product intermediate: MF0.2 μm filtered', and samples were collected for in-process bioburden testing. The material was stored overnight at 2 ℃ to 8 ℃ for further processing. An additional filtration step may be performed on the day of chromatography prior to applying the clarified cell lysate to the chromatography column.

8.3.2.4 purification by anion exchange chromatography

The sodium chloride concentration of the 0.2 μm filtered product intermediate was adjusted by adding dilution buffer AAV8. The cell lysate containing the carrier is purified by ion exchange chromatography using ion exchange resin. The GE Healthcare AKTA pilot chromatography system was equipped with a BPG column containing about 1L resin bed volume. The chromatographic column employs continuous flow conditions and meets established asymmetric specifications. The system was sterilized according to the established procedure and stored in 20% ethanol until the next run. Prior to use, the buffer equilibration system was washed with sterile AAV8. Using aseptic techniques and sterile materials and components, BPC containing clarified cell lysate was connected to the sterilized sample inlet port, and BPC listed below containing biological treatment buffer was connected to the sterilized inlet port of the AKTA assay. All the connections in the chromatographic process are performed aseptically. The cleared cell lysate was applied to a chromatography column and washed with AAV8 wash buffer. Under these conditions, the support is bound to the column and the impurities are washed out of the resin. AAV8 particles were eluted from the column by AAV8 elution buffer and collected in sterile plastic bottles. The material was labeled as 'product intermediate' and samples were collected for in-process bioburden testing. The material is immediately further processed.

8.3.2.5 CsCl gradient ultracentrifugation purification

AAV8 particles purified by anion exchange column chromatography as described above contain empty capsids and other product-related impurities. Empty capsids were separated from the carrier particles by cesium chloride gradient ultracentrifugation. Using aseptic technique, cesium chloride was added to the carrier "product intermediate" and gently mixed to a final concentration of 1.35g/mL density. The solution was filtered through a 0.2 μm filter, distributed into ultracentrifuge tubes, and ultracentrifuged in a 15 ℃ Ti50 rotor for about 24 hours. After centrifugation, the ultracentrifuge tube was removed from the rotor, wiped with Septiohol, and placed in the BSC. Each ultracentrifuge tube is clamped on a rack and is illuminated in focus to aid visualization of the bands, typically with the two major bands being observed, the upper band corresponding to the empty capsids and the lower band corresponding to the carrier particles. The lower band was recovered from each ultracentrifuge tube and a sterile needle attached to a sterile syringe was used. The recovered carriers from each ultracentrifuge tube were combined and samples were collected to determine in-process bioburden, endotoxin and carrier titer. The pooled material was distributed into sterile 50mL polypropylene conical tubes labeled' product intermediate: CsCl gradient followed', and stored immediately at-80 ℃ until the next process step.

8.3.2.6 buffer exchange by tangential flow filtration

After testing and release for pooling, multiple batches of the vector purified by CsCl band process steps were combined and diafiltered by TFF to produce large quantities of vector. The volume of the pooled carrier was adjusted according to the titer of the samples obtained from the individual batches using the calculated volume of sterile diafiltration buffer. Depending on the volume available, aliquots of concentrated, concentration-adjusted carriers are single-use TFF devices. The apparatus is sterilized prior to use and then equilibrated in diafiltration buffer. Once the filtration process is complete, the support is recovered from the TFF apparatus and placed in a sterile vial. This material was labeled "pre-0.2 μm filter". The material is immediately further processed.

8.3.2.7 formulation and 0.2 μm filtration to prepare bulk carriers

Batches prepared from individual TFF units were pooled together and gently swirled in 500mL sterile vials. Then, a large amount of the carrier was prepared through a 0.22 μm filter. The pooled material was sampled for bulk carrier and retained QC testing and then introduced into a 50mL sterile polypropylene tube, labeled "bulk carrier" and stored at-80 ℃ until the next step.

8.4. Carrier testing

A characterization analysis was performed that included serotype identity, empty particle content, and transgenic product identity. A description of all analyses is presented below.

8.4.1 Genomic Copy (GC) titres

Genome copy titers were determined by comparison to homologous plasmid standards using an optimized quantitative pcr (oqpcr) method. oqPCR analysis sequential cleavage with DNase I and Proteinase K followed by qPCR analysis to determine vector genome copies. DNA detection is performed by targeting the RBG polyA region with sequence specific primers and hybridizing the region with a fluorescently labeled probe. Comparison with a plasmid DNA standard curve allows for titer determination without any post-PCR sample manipulation. Many standards, validation samples and controls (background and DNA contamination) have been introduced into the analysis. The assay method has been identified by establishing and defining analytical parameters including sensitivity, detection limits, qualitative range, and precision within and between assays. Internal AAV8 reference batches were established and used to perform validation studies.

8.4.2 potency assay

In vivo potency assays were designed to detect human LDLR vector-mediated reduction of total cholesterol levels in a Double Knockout (DKO) LDLR-/-Apobec-/-mouse model of HoFH. The basis of the in vivo potency assay is described in section 4.3.5.11. To determine the efficacy of aav8.tbg. hldlr, old DKO mice 6 to 20 weeks old were injected intravenously (tail vein) 5 × 10 per mouse¹¹GC/kg of vector diluted in PBS. Animals were bled by retroorbital bleeding and serum total cholesterol levels assessed by Antech GLP before and after vehicle administration (day 14 and day 30). From past experience with vehicle administration at this dose, it is expected that the total cholesterol level will drop by 25% to 75% in animals administered the vehicle by day 14. According to the expected range of total cholesterol reduction, 5X 10 per mouse was selected in clinical trials¹¹GC/kg dose, which will allow assessment of changes in carrier potency during stability testing.

8.4.3 vector capsid identity: AAV capsid mass spectrometry analysis of VP3

Analysis of the VP3 capsid protein polypeptide by Mass Spectrometry (MS) identified AAV2/8 serotype of the vector. The method involves multienzyme cleavage of the VP3 protein band (trypsin, chymotrypsin and endoproteinase Glu-C) from SDS-PAGE gels followed by characterization by UPLC-MS/MS on a Q-extraction orbitrap mass spectrometer to rank the capsid proteins. A tandem Mass Spectrometry (MS) method was developed that allowed the identification of certain contaminating proteins and the acquisition of peptide sequences from mass spectra.

8.4.4 empty to full particle ratio

Sedimentation velocity measured by Analytical Ultracentrifuge (AUC) is a good way to obtain information on the heterogeneity of macromolecular structures, confirmation of differences and association or aggregation status. The samples were loaded into the cell and pelleted in a Beckmann Coulter proteins laboratory XL-I analytical ultracentrifuge at 12000 RPM. Refractive index scans were recorded every two minutes for 3.3 hours. The data were analyzed by a c(s) model (Sedfit program) to calculate the deposition coefficient corresponding to the normalized c(s) value. A major peak representing a single support should be observed. The appearance of a peak with a slower migration rate than the peak of the main monomer indicates empty/misassembled particles. The sedimentation coefficient of the empty particle peak was established using the empty AAV8 particle formulation. The dominant monomer peak and direct quantification of the aforementioned peaks can determine the empty to full particle ratio.

8.4.5 infectious titer

Infection Unit (IU) assays were used to determine productive uptake and replication of the vector in RC32 cells (rep 2 expressing HeLa cells). Briefly, RC32 cells in 96-well plates were co-infected by serial dilution of vector and uniform dilution of Ad5, with 12 replicates per dilution of rAAV. Cells are cracked after seventy-two hours of infection, and the rAAV vector is detected to be amplified after inputting by qPCR. Replication titers were determined in IU/ml using endpoint dilution TCID50 calculation (Spearman-Karber). Since the "infectivity" value depends on the particle in contact with the cell, receptor binding, internalization, transport to the nucleus and genomic replication, it is influenced by the assay geometry and the presence of the appropriate receptor and post-binding pathways in the cell line used. Receptor and post-binding pathways critical for AAV vector import are usually maintained in immortalized cell lines, and thus infectivity assay titers are not an absolute measure of the number of "infectious" particles. However, the ratio of GC to "infectious units" of protein capsid encapsulation (referred to as GC/IU ratio) can be used to measure the product consistency of each batch. Due to the low infectivity of AAV8 vector in vitro, the variability of this in vitro bioassay may be high (30% to 60% CV).

8.4.6 transgene expression analysis

At the receiving of the slave1×10¹⁰GC(5×10¹¹GC/kg) of aav8.tbg. hldlr vector in LDLR-/-Apobec-/-mice harvested liver. Animals dosed 30 days ago with vehicle were euthanized, livers harvested and homogenized in RIPA buffer. Whole liver homogenates of 25ug to 100ug were run on 4% to 12% denaturing SDS-PAGE gels and detected using anti-human LDLR antibodies to determine transgene expression. Animals that did not receive a vector or an unrelated vector were used as controls for the experiment. The vector treated animals are expected to show a band migrating in the 90-160kDa range due to post-translational modifications. The relative expression level was determined by quantifying the integrated intensity of the bands.

(sequence listing free text)

For sequences containing free text under the numeric identifier <223>, the following information is provided.

All publications cited in this specification are herein incorporated by reference in their entirety, as in U.S. provisional patent application No. 62/782,627, filed on 12/20/2018. Similarly, SEQ ID NO referenced herein and appearing in the additional sequence list, labeled "16-7717C 2PCT _20191210_ sequence listing _ ST 25", dated 12, month 10, 2019, is 64,936 bytes in size, and is incorporated herein by reference. Although the invention has been described with reference to specific embodiments, it will be understood that modifications may be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

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