Composition for treating spinal muscular atrophy
阅读说明：本技术 用于治疗脊髓性肌萎缩的组合物 (Composition for treating spinal muscular atrophy ) 是由 J.M.威尔逊 C.辛德勒 N.卡茨 王强 于 2018-02-27 设计创作，主要内容包括：本文描述了具有AAVhu68衣壳和所述衣壳中的至少一个表达盒的rAAV载体。所述至少一个表达盒包含编码功能性SMN蛋白的核酸序列和指导SMN序列在宿主细胞中表达的表达控制序列。还提供了含有此rAAVhu68.SMN载体的组合物以及将所述组合物用于患者的脊髓性肌萎缩的方法。(Described herein are rAAV vectors having an AAVhu68 capsid and at least expression cassettes in the capsid, the at least expression cassettes comprise a nucleic acid sequence encoding a functional SMN protein and an expression control sequence that directs expression of the SMN sequence in a host cell, compositions containing such raavhu68.SMN vectors, and methods of using the compositions for spinal muscular atrophy in a patient.)
A recombinant adeno-associated virus (rAAV) vector comprising an AAVhu68 capsid and at least expression cassettes, wherein the at least expression cassettes comprise a nucleic acid sequence encoding a functional SMN protein and expression control sequences that direct expression of the SMN sequence in a host cell, wherein the rAAV has an AAVhu68 capsid, the AAVhu68 capsid comprising:
a heterogeneous population of AAVhu68 vp1 protein, said AAVhu68 vp1 protein selected from the group consisting of a vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO. 8 from 1 to 736, a vp1 protein produced by SEQ ID NO. 7, or a vp1 protein produced by a nucleic acid sequence having at least 70% identity with SEQ ID NO. 7 encoding the predicted amino acid sequence of SEQ ID NO. 8 from 1 to 736,
a heterogeneous population of AAVhu68 vp2 proteins, said AAVhu68 vp2 protein selected from the group consisting of a vp2 protein produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO. 8, a vp2 protein produced by a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO. 7, or a vp2 protein produced by a nucleic acid sequence having at least 70% identity with at least nucleotides 412 to 2211 of SEQ ID NO. 7 encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO. 8,
a heterogeneous population of AAVhu68 vp3 proteins, said AAVhu68 vp3 protein selected from the group consisting of a vp3 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID No. 8, a vp3 protein produced by a sequence comprising at least nucleotides 607 to 2211 of SEQ ID No. 7, or a vp3 protein produced by a nucleic acid sequence having at least 70% identity with at least nucleotides 607 to 2211 of SEQ ID No. 7 encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID No. 8.
2. The rAAV vector according to claim 1, wherein the encoded SMN protein is an isoform D protein.
3. The rAAV vector according to claim 1 or 2, wherein the encoded SMN isoform D protein has the amino acid sequence of SEQ id No. 2.
4. The rAAV vector according to any of claims 1 to 3, wherein the nucleic acid sequence encoding SMN isoform D is selected from the group consisting of:
(a) 1 or a sequence sharing at least 70% identity therewith encoding an SMN protein having the amino acid sequence of SEQ ID No. 2;
(b)SEQ ID NO:4；
(c) 5 or SEQ ID NO
(d)SEQ ID NO:6。
5. The rAAV vector of of any one of claims 1 to 4, wherein the expression control sequence comprises a promoter.
6. The rAAV vector according to claim 5, wherein the promoter is a chicken β -actin (CB) promoter.
7. The rAAV vector according to claim 6, wherein the promoter is a CB7 promoter.
8. The rAAV vector of of any one of claims 1 to 7, further comprising or more of an enhancer, an intron, a Kozak sequence, a poly A, a post-transcriptional regulatory element.
9. The AAV vector of any one of claims 1-8, , further comprising an AAV Inverted Terminal Repeat (ITR) sequence from a different AAV than the AAV providing the capsid.
10. The AAV vector of claim 9, wherein the ITRs are from AAV 2.
An recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid that packages in the capsid a vector genome comprising an AAV 5 'ITR, a CB7 promoter, an intron, the nucleic acid sequence of SEQ ID NO:1, a poly A, and an AAV 3' inverted terminal repeat.
12. The rAAV of claim 11, wherein the vector genome has the sequence of SEQ ID NO 15 or SEQ ID NO 25.
13. The rAAV according to claim 11 or 12, wherein the AAV has an AAVhu68 capsid comprising: wherein the AAVhu68 capsid comprises:
a heterogeneous population of AAVhu68 vp1 protein, said AAVhu68 vp1 protein selected from the group consisting of a vp1 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO. 8 from 1 to 736, a vp1 protein produced by SEQ ID NO. 7, or a vp1 protein produced by a nucleic acid sequence having at least 70% identity with SEQ ID NO. 7 encoding the predicted amino acid sequence of SEQ ID NO. 8 from 1 to 736,
a heterogeneous population of AAVhu68 vp2 proteins, said AAVhu68 vp2 protein selected from the group consisting of a vp2 protein produced by expression of a nucleic acid sequence encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO. 8, a vp2 protein produced by a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO. 7, or a vp2 protein produced by a nucleic acid sequence having at least 70% identity with at least nucleotides 412 to 2211 of SEQ ID NO. 7 encoding a predicted amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO. 8,
a heterogeneous population of AAVhu68 vp3 proteins, said AAVhu68 vp3 protein selected from the group consisting of a vp3 protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID No. 8, a vp3 protein produced by a sequence comprising at least nucleotides 607 to 2211 of SEQ ID No. 7, or a vp3 protein produced by a nucleic acid sequence having at least 70% identity with at least nucleotides 607 to 2211 of SEQ ID No. 7 encoding the predicted amino acid sequence of at least about amino acids 203 to 736 of SEQ ID No. 8.
14. The rAAV according to claim 13, wherein the AAVhu68 is further characterized as having a valine at position 157 and optionally a glutamic acid at position 67.
15. The adeno-associated virus (AAV) vector according to any of claims 1-14, wherein AAV is for use in treating spinal muscular atrophy in a patient.
16, a pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, and/or preservative, and the AAV vector of any of claims 1-15.
A method of treating spinal muscular atrophy in a subject, the method comprising administering the composition of claim 16 to a subject in need thereof.
18. The method of claim 17, wherein the composition is administered intrathecally.
19. The method of claim 17 or 18, wherein the subject is a mammal.
20. The method of of any one of claims 17-19, wherein the subject is a human.
21. The method of of any one of claims 17-20, wherein the composition is administered in combination with another therapies.
22. The method of of any one of claims 17 to 21, wherein the composition is at about 1x1010GC/g brain mass to about 3x1014GC per gram brain mass dose administration.
23. The method of claim 22, wherein the composition is administered at about 5x1013Dosage of GC.
24. The method of claim 22, wherein the composition is administered at about 1.85x1014Dosage of GC.
25. The method of any of claims 17 to 24, wherein the composition is administered more than times.
26. The rAAV according to any in claims 1-15 or the composition according to claim 16 for use in treating spinal muscular atrophy, the method comprising administering the composition of claim 16 to a patient in need thereof.
27. The rAAV according to claim 26, wherein the rAAV is in a composition formulated for intrathecal delivery.
28. The rAAV according to claim 26 or 27, wherein the rAAV is co-administered in combination with another therapies.
29. The rAAV according to any of claims 26 to 28, wherein the rAAV is at about 1x1010GC/g brain mass to about 3x1014GC/g brain mass or about 1.85x1014GC or about 5x1013Dosage of GC.
30. The rAAV according to any of claim 26 to claim 29, wherein the rAAV is suspended in an aqueous solution at a pH of 7.2 to 7.8.
31. Use of the rAAV according to in any of claims 1 to 15 or the composition according to claim 16.
32. The use of claim 31, wherein the rAAV is in a composition formulated for intrathecal delivery.
33. The use of claim 31 or 32, wherein the rAAV is co-administered in combination with another therapies.
34. The use of in any one of claims 31-33, wherein the rAAV is administered at about 1x1010GC/g brain mass to about 3x1014GC/g brain mass or about 1.85x1014GC or about 5x1013Dosage of GC.
35. The use of of any one of claims 31-34, wherein the rAAV is suspended in an aqueous solution having a pH of 7.2 to 7.8.
36. The use of of any one of claims 31-35, for treating spinal muscular atrophy in children under 18 years of age.
37. The use of of any one of claims 31-35, for treating spinal muscular atrophy in an adult.
Background
Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in telomeric SMN1, and SMN1 is a gene encoding a ubiquitously expressed protein involved in spliceosome biogenesis (a survivor of motor neuron, SMN). SMA is an autosomal recessive genetic disease caused by a mutation or deletion in the SMN1 gene. Providing a functional SMN1 gene has been shown to rescue this phenotype. See, for example, Tanguy et al, System AAVrh10 videos high gene expression can be AAV9 in the woven and the vertebral cord of the neural die, Frontiers in Molecular neural, 8(36) (7 months 2015).
The International SMA association classification (The International SMA Consortium classification) defines multiple severity levels of SMA phenotype based on age of onset and motor development milestones. SMA0 is named to reflect prenatal episodes and severe joint contractures, facial diploplegia, and respiratory failure. SMA type 1 (or I) (wedneshi-hofmann disease I) is the most severe postpartum form, with onset within 6 months after birth. The patient is unable to sit up and has severe respiratory function disorders. Type 2 (or II) SMA is an intermediate form that is diseased within the first 2 years; the child can sit up but cannot walk. The clinical course is variable. Type 3 (or III), also known as kugelberg-welan disease, begins after 2 years of age and usually has a chronic progression. The child may stand and walk independently, at least during infancy. Adult form (
Many genetic and acquired neuromuscular diseases involve degeneration of the lower motor neuron (SMA) the most common and devastating example is Spinal Muscular Atrophy (SMA), which is a genetic defect in the motor neuron Survival (SMN) protein, characterized by selective death of the lower motor neuron, progressive weakness, and death usually early in childhood.
The clear correlation between SMN expression and disease severity and the relatively small number of affected cells make SMA an excellent target for gene therapy. Previous studies have demonstrated that SMA phenotypes can be rescued in transgenic mouse models using systemic injection of AAV vector serotypes that are capable of crossing the blood-brain barrier. See, for example, Tangguy et al, systemicAAVrh10 videos high gene expression than AAV9 in the clone and the protein code of the neural microorganism, Frontiers in Molecular Neuroscience,8(36) (7 months 2015). Passsini et al, HGT,2014 reported a dose-dependent increase in survival in
There remains a need for effective treatments for SMA.
Disclosure of Invention
In aspects, recombinant adeno-associated virus (rAAV) vectors are provided comprising an AAVhu68 capsid and at least expression cassettes, wherein the at least expression cassettes comprise a nucleic acid sequence encoding a functional SMN protein and an expression control sequence that directs expression of the SMN sequence in a host cell, wherein the AAVhu68 capsid comprises a population of AAVhu68vp1 capsid proteins, the AAVhu68vp1 capsid proteins having an amino acid sequence independently selected from the group consisting of the proteins encoded by SEQ ID NO:7, or having an amino acid sequence of SEQ ID NO:8 in certain embodiments, the AAVhu68 capsid has a heterogeneous population of vp1 proteins in certain embodiments, the AAVhu68 has a heterogeneous population of vp2 proteins in certain embodiments, the AAVhu68 capsid has a heterogeneous population of vp3 proteins in embodiments, the AAVhu68 has the sequence of the SEQ ID SMA protein in .
Also provided are rAAV vectors comprising an AAVhu68 capsid, the AAVhu68 capsid packaging in the capsid a nucleic acid molecule comprising an AAV5 'ITR, a CB7 promoter, an intron, a nucleic acid sequence of SEQ ID NO:1, a poly a, and an AAV 3' inverted terminal repeat, wherein the AAVhu68 capsid comprises a population of vp1 proteins, a population of vp2, and a population of vp3 proteins, wherein the AAVhu68 capsid protein has an amino acid sequence independently selected from the group consisting of proteins produced by SEQ ID NO:7, the capsid protein having an amino acid sequence of vp1, vp2, and/or vp3 of SEQ ID NO: 8. In certain embodiments, the AAVhu68 capsid has a heterogeneous population of vp1 proteins. In certain embodiments, the AAVhu68 capsid has a heterogeneous population of vp2 proteins. In certain embodiments, the AAVhu68 capsid has a heterogeneous population of vp3 proteins.
Pharmaceutical compositions comprising the AAVhu68 vector as described above are provided the compositions further comprise at least pharmaceutically acceptable carriers, excipients, and/or preservatives in addition to at least carrier stock solutions (vector stock).
Also provided are methods of treating spinal muscular atrophy in a subject in need thereof using raavhu68.sma vectors or other delivery vehicles for the engineered hSMN provided herein. In certain embodiments, the compositions provided herein can be administered intrathecally.
In certain embodiments, raavhu68.smn1 or a composition as described herein is provided for use in treating spinal muscular atrophy in a patient, optionally in co-therapy. In certain embodiments, the patient has
Use of raavhu68.SMA or a composition comprising the same as described herein for treating a patient having SMA (optionally in a co-treatment regimen) is provided. Such compositions can be formulated for intrathecal delivery.
Other aspects and advantages of the invention will become apparent from the following detailed description of the invention.
Drawings
FIG. 1A is a schematic representation of the AAVhu68.CB7.CI. hSMN1co. RBG vector genome, ITR stands for AAV2 inverted terminal repeat, CB7 stands for chicken β actin promoter with cytomegalovirus enhancer, RBG poly A stands for rabbit β globin polyadenylation signal.
FIGS. 1B-1C are alignments of native hSMN1, variant D (accession NM-000344.3) (SEQ ID NO:3) and the codon optimized sequence described herein (SEQ ID NO: 1).
Fig. 2 is an assessment of transduction in brain and spinal cord by Immunohistochemistry (IHC) and In Situ Hybridization (ISH). Immunohistochemistry for SMN1 was performed on cortical, cerebellar, and spinal cord sections, and representative images are shown in the top three panels. ISH of codon optimized hSMN1 ribonucleic acid (RNA) was performed in cortical sections and representative results are shown in the bottom panel. From with or without 3x1010GC/pup aavhu68.cb7.ci. hsmn1.rbg treated SMN Δ 7(KO) mice samples were collected. Wild Type (WT) and heterozygous (Het) littermates were used as positive controls.
FIG. 3A shows the use or non-use of 3x1010Or 8.76x1010Survival curves of GC/pup aavhhu 68.cb7.ci. hsmn1.rbg treated SMN Δ 7(KO) mice. Wild Type (WT)/heterozygous (Het) littermates and PBS injected mice were used as controls.
Fig. 3B is a table of statistical analysis of the survival curves depicted in fig. 3A. Median survival was calculated and listed.
FIG. 4A shows a graph of 3X1010Or 8.76x1010Line graph of body weight of GC/pup aavhu68.cb7.ci. hs mn1.rbg vector treated SMN Δ 7(KO) mice. Wild Type (WT)/heterozygous (Het) littermates and PBS injected mice were used as controls.
FIG. 4B shows a schematic representation of the 3X1010Or 8.76x1010GC/pup body weight plots of aavhhu 68.cb7.ci. hs mn1.rbg treated SMN Δ 7(KO) mice on
FIG. 4C shows a schematic representation of the 3X1010Or 8.76x1010GC/pup weight plots of aavhhu 68.cb7.ci. hs mn1.rbg treated SMN Δ 7(KO) mice on
FIGS. 4D-4J are line graphs of body weight after gender stratification versus 3X1010Or 8.76x1010GC/pup aavhhu 68.cb7.ci. hsmn1.rbg treated SMN Δ 7(KO) mice, experiments were performed every two days from
FIG. 5A is a graphical representation of a scoring system used in the Hind Limb Suspension Test (Hind-Limb Suspension Test).
FIG. 5B includes a table for the user 3x1010Or 8.76x1010Graph of hindlimb scores recorded every two days from
Fig. 6A-6C include graphs showing the time it takes for an animal to return to its normal position in a Righting Reflex Test. For the use of 3x1010Or 8.76x1010GC/pup aavhhu 68.cb7.ci. hsmn1.rbg treated SMN Δ 7(KO) mice, experiments were performed every two days from
FIGS. 6D-6J are pictorial representations showing differentiation at by genderAnd then the time it takes for the animal to return to its normal position in a righting reflex test. For the use of 3x1010Or 8.76x1010GC/pup aavhhu 68.cb7.ci. hsmn1.rbg treated SMN Δ 7(KO) mice, experiments were performed every two days from
Fig. 7 is an image of a device for intracisternal delivery including an optional introducer needle for a coaxial insertion method, the device including a 10cc carrier syringe, a 10cc pre-filled flush syringe, a T-connector extension set, a 22Gx5 "spinal needle, an optional 18gx3.5" introducer needle.
Figure 8A provides an alignment showing the amino acid sequence of the vp1 capsid sequence encoded by the nucleic acid sequences of figures 8B-8D. The alignment included the following vp1 proteins: AAVhu68[ SEQ ID NO:8] was compared to AAV9[ SEQ ID NO:16], AAVhu31 (labeled hu.31 in the alignment) [ SEQ ID NO:18] and AAVhu32 (labeled hu.32 in the alignment) [ SEQ ID NO:19 ]. In contrast to AAV9, AAVhu31, and AAVhu32, the discovery of two mutations in AAVhu68 (a67E and a157V) is critical and is circled in the figure.
Fig. 8B-8D provide an alignment of nucleic acid sequences encoding the vp1 capsid: AAVhu68[ SEQ ID NO:7] was compared with AAV9[ SEQ ID NO:22], AAVhu31[ SEQ ID NO:20] and AAVhu32[ SEQ ID NO:21 ].
FIGS. 9A-9B are graphs (1X 10) showing the results of weight monitoring in adult wild-type mice (HET/WT, FIG. 9A) or
FIGS. 10A-10B are graphs (1x 10) showing the results of orthotropic reflexes in adult wild type mice (HET/WT, FIG. 10A) or
Fig. 11A-11H are graphs showing clinical pathology, CSF chemistry and CSF cytology of adult rhesus monkeys treated intrathecally with aavhu68.cb7.ci. hsmn1co. rbg as described in example 10.
Figures 12A-12B provide quantification of motor neuron transduction by ISH (figure 12A) and IHC (figure 12B) in adult rhesus monkeys treated intrathecally with aavhu68 cb7 ci hsmn1co rbg as described in example 10. confirmation of placement of needles in the cisterna magna (intra cisterna magna); ICM) or lumbar cisterna (lumbar puncture; LP) using fluoroscopic guidance and contrast material total injection volume of 1.0mL for ICM (n ═ 3) and 2.5mL (n ═ 4) or 5.0mL (n ═ 4) for LP group months post injection, animals were sacrificed, and motor neuron transduction was quantified by Immunohistochemistry (ISH) of transgenic mRNA and Immunohistochemistry (IHC) of human SMN protein in spinal cord sections.
Figure 13 provides biodistribution in adult rhesus monkeys treated intrathecally with aavhu68.cb7.ci. hsmn1.rbg as described in example 10.
Figures 14A-14B provide results of body weight monitoring of adult C57BL/6J mice treated with aavhu68.cb7.ci. hsmn1co. rbgicv as described in example 9. Figure 14A provides the results of weight monitoring of female subjects. Figure 14B provides the results of weight monitoring of male subjects.
Fig. 15A-15B are flow charts of the manufacture of aavhu68.smn vectors.
Figures 16A-16E provide graphs showing an increase in acute transaminases following intravenous administration of AAV vectors expressing human SMN to non-human primates. Figure 16A provides the study design as described in
Figures 17A-17D provide representative IHC images showing liver histopathological findings for animal 16C176 a number of acute hepatocellular necrosis (figure 17A) and sinuous fibrin deposition in veins (figure 17B, arrows) and acute fibrin thrombus (figure 17C) (hematoxylin and eosin;
Figures 18A-18D provide representative neurological
Figure 19 provides vector biodistribution in rhesus monkeys. Rhesus monkeys treated intravenously with AAVhu68 expressing human SMN were euthanized 28 days post-injection, except for animal 16C176, which developed liver failure and
FIGS. 20A-20G show SMN expression in rhesus monkeys, and human SMN RNA was detected by ISH in the liver (FIG. 20A). The livers were stained with control probes for albumin (fig. 20B) and GFP (fig. 20C). SMN-expressing cells were identified by ISH in the spinal cord (fig. 20D). Motor neurons were identified by ChAT ISH (fig. 20E). Sparse patches of transduced neurons detected by SMN ISH in brain (fig. 20F, DAPI nuclear staining). The percentage of transduced ChAT + motor neurons at each spinal cord level was quantified (fig. 20G). Error bar-SEM.
Figures 21A-21D provide representative histopathological findings of piglets treated intravenously with AAVhu68 expressing human SMN at 7 and 30 days of age axons were observed for dorsal white matter tracts in both groups overall (figure 21A) dorsal axonal lesions were bilateral and characterized by dilated myelin sheath with and without bone marrow macrophages, with axonal degeneration caused by axonal degeneration dorsal root ganglia of the spinal cord (figure 21B) exhibited varying degrees of neuronal cell somatosis characterized by central nismus lysis, satellite status and mononuclear cell infiltration surrounding and invading neuronal cell bodies (neurophagocytosis) in most piglets, different degrees of similar axonal lesions were observed in peripheral nerves of hind (sciatic nerve, figure 21C) and forelimb (median nerve, figure 21D) (hematoxylin and eosin;
Figure 22 provides vector biodistribution in piglets. Newborn piglets treated intravenously with AAVhu68 expressing human SMN were euthanized 13-14 days after injection. Vector genomes were detected in tissue DNA samples by quantitative PCR. Values are expressed as vector Genome Copies (GC) per diploid host genome. DRG ═ dorsal root ganglia. Data for four liver lobes (caudate, left, middle and right) are shown.
Fig. 23A-23F provide representative images showing SMN expression in the spinal cord of piglets. Human SMN RNA was detected by ISH in cervical (fig. 23A), thoracic (fig. 23B) and lumbar (fig. 23C) spinal segments. Motor neurons were identified by ChAT ISH in the corresponding sections (fig. 23D-23F). A representative image is displayed.
Detailed Description
Provided herein are recombinant AAVhu68 vectors having an AAVhu68 capsid and a nucleic acid encoding a motor neuron Survival (SMN) gene under the control of regulatory sequences directing its expression in a patient in need thereof, rAAVhu68 capsid comprises proteins independently having the amino acid sequence produced by SEQ ID NO:7 and/or having the amino acid sequence of SEQ ID NO:8 compositions comprising these vectors are provided, and the use of these vectors in compositions for treating SMA patients.
I.AAV
As used herein, the term "clade" in relation to the AAV group refers to groups of AAV that are phylogenetically related to each other, as determined by at least 75% bootstrap value (at least 1000 repeats) using the Neighbor-Joining algorithm and a Poisson correction distance (Poisson correction distance) measurement of no greater than 0.05 based on AAV vp1 amino acid sequence alignment, the Neighbor-Joining algorithm has been described in the literature, see, e.g., M.Nei and S.Kumar, Molecular Evolution Phytolenes (Oxford university Press, New York (2000) computer programs useful for implementing the algorithm are available.
As used herein, an "
AAVhu68 is composed of an AAVhu68 capsid and a Vector genome AAVhu68 capsid is an assembly of a heterogeneous population of vp1, a heterogeneous population of vp2 and a heterogeneous population of vp3 protein as used herein, the term "heterogeneous" or any grammatical variant thereof, when used to refer to a vp capsid protein, refers to a population composed of non-identical elements, such as vp1, vp2 or vp3 monomers (proteins) having different modified amino acid sequences,
The AAVhu68 capsid comprises subgroups within the vp1 protein, within the vp2 protein and within the vp3 protein with modifications relative to the predicted amino acid residues in SEQ ID No. 8 these subgroups comprise at least some deamidated asparagine (N or Asn) residues.
As used herein, unless otherwise specified, a "subset" of vp proteins refers to a group of vp proteins that have at least defined common characteristics, and consist of at least group members to less than all members of a reference group.
Unless otherwise specified, highly deamidated means at least 45% deamidation, at least 50% deamidation, at least 60% deamidation, at least 65% deamidation, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 97%, 99%, up to about 100% deamidation at a reference amino acid position compared to the predicted amino acid sequence at the reference amino acid position (e.g., at least 80% of the asparagines at amino acid 57 of SEQ ID NO:8 can be deamidated based on the total vp1 protein, or 20% of the asparagines at amino acid 409 of SEQ ID NO:8 can be deamidated based on the total vp1, vp2, and vp3 proteins). Such percentages may be determined using 2D-gel, mass spectrometry techniques, or other suitable techniques.
Without wishing to be bound by theory, deamidation of at least the highly deamidated residues in the VP protein in the AAVhu68 capsid is believed to be primarily non-enzymatic in nature, caused by groups in the capsid protein that deamidate selected asparagine and to a lesser extent glutamine residues.efficient capsid assembly of most deamidated VP1 proteins suggests that these events occur after capsid assembly, or deamidation in individual monomers (VP1, VP2 or VP3) is structurally well tolerated and does not affect the assembly kinetics to a large extent. panteamidation in the VP 1-unique (VP1-u) region (-aa 1-137) is generally believed to be located inside prior to cell entry, suggesting that VP deamidation may occur prior to capsid assembly.
Without wishing to be bound by theory, deamidation of N can occur by nucleophilic attack of the main chain nitrogen atom of its C-terminal residue on the side chain amide group carbon atom of Asn. Intermediate ring-closed succinimide residues are believed to be formed. The succinimide residue is then subjected to rapid hydrolysis to give the final product aspartic acid (Asp) or isoaspartic acid (IsoAsp). Thus, in certain embodiments, deamidation of asparagine (N or Asn) produces Asp or IsoAsp, which can be interconverted through a succinimide intermediate, for example as shown below.
As referred to herein, each deamidated N of SEQ ID NO:8 can independently be aspartic acid (Asp), isoaspartic acid (isoAsp), aspartate (asparate), and/or a tautomeric mixture of Asp and isoAsp, or combinations thereof, any suitable ratio of α -to isoaspartic acid can be present, for example, in certain embodiments, the ratio can be from 10:1 to 1:10 aspartic acid to isoaspartic acid, about 50:50 aspartic acid to isoaspartic acid, or about 1:3 aspartic acid to isoaspartic acid, or other selected ratios.
In certain embodiments, or more glutamines (Q) in SEQ ID NO:8 are deamidated to glutamic acid (Glu), i.e., α -glutamic acid, gamma-glutamic acid (Glu), or a mixture of α -and gamma-glutamic acid, which can be interconverted through a common glutarimide (glutarinimide) intermediate, α -and gamma-glutamic acid can be present in any suitable ratio, for example, in certain embodiments, the ratio can be α to gamma of 10:1 to 1:10, α to gamma of about 50:50, or α to gamma of about 1:3, or other selected ratios.
Thus, rAAVhu68 comprises within the rAAVhu68 capsid a subset of the vp1, vp2, and/or vp3 proteins with deamidated amino acids that comprises at least a subset of comprising at least highly deamidated asparagines.
In certain embodiments, the AAVhu68 capsid comprises subpopulations of vp1, vp2, and vp3 having at least 4 to at least about 25 deamidated amino acid residue positions as compared to the encoding amino acid sequence of SEQ ID No. 8, wherein at least 1% to 10% are deamidated. Most of these may be N residues. However, the Q residue may also be deamidated.
In certain embodiments, the AAV68 capsid is further characterized by or more of the AAV hu68 capsid proteins comprising the AAVhu68vp1 protein resulting from expression of the nucleic acid sequence encoding the predicted amino acid sequence from 1 to 736 of SEQ ID NO:8, the vp1 protein resulting from SEQ ID NO:7, or the vp1 protein resulting from a nucleic acid sequence having at least 70% identity to with SEQ ID NO:7 encoding the predicted amino acid sequence from 1 to 736 of SEQ ID NO:8, the AAVhu68 vp2 protein resulting from expression of the nucleic acid sequence encoding the predicted amino acid sequence from at least about amino acids 138 to 736 of SEQ ID NO:8, the vp2 protein resulting from a sequence comprising at least nucleotides 412 to 2211 of SEQ ID NO:7, or the vp2 protein resulting from a nucleic acid sequence having at least 70% identity to 2211 with at least about nucleotide sequence of SEQ ID NO:7 encoding the predicted amino acid sequence from at least about amino acids 138 to 736 of SEQ ID NO:8, or the nucleic acid sequence of the vp 898 protein resulting from the predicted amino acids 96203 protein resulting from the nucleic acid sequence encoding the amino acids of SEQ ID NO: 203 to 2211, or the predicted amino acids of SEQ ID NO:8, the protein resulting from the nucleotide sequence comprising at least about vhu 9612 to 2211, at least about vhu 9670, at least about v898, the predicted amino acids of SEQ ID No. equivalent to 2211, or at least about v7.
Additionally or alternatively, AAV capsids are provided, based on the numbering of the vp1 capsid of SEQ ID NO:8, comprising a heterogeneous population of vp1 protein, a heterogeneous population of vp2 protein (optionally comprising a valine at position 157) and a heterogeneous population of vp3 protein, wherein at least the vp1 and vp2 subpopulations comprise a valine at position 157 and optionally further comprise a glutamic acid at position 67 additionally or alternatively AAVhu68 capsids are provided comprising a heterogeneous population of vp1 protein which is the product of a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:8, a heterogeneous population of vp2 protein which is the product of a nucleic acid sequence encoding an amino acid sequence of at least about amino acids 138 to 736 of SEQ ID NO:8, and a heterogeneous population of vp3 protein which is the product of a nucleic acid sequence encoding at least
The AAVhu68vp1, vp2 and vp3 proteins are usually expressed as alternative splice variants encoded by the same nucleic acid sequence encoding the full-length vp1 amino acid sequence (
As described herein, rAAVhu68 has rAAVhu68 capsids generated in a production system from an AAVhu68 nucleic acid encoding the vp1 amino acid sequence of SEQ ID NO:8 and optionally additional nucleic acid sequences, e.g., encoding vp3 proteins that do not contain the vp1 and/or vp2 unique regions. rAAVhu68 generated using a single nucleic acid sequence vp1 produced a heterogeneous population of vp1 protein, vp2 protein and vp3 protein. More specifically, the AAVhu68 capsid comprises subgroups within the vp1 protein, within the vp2 protein and within the vp3 protein with modifications relative to the predicted amino acid residues in seq id No. 8. These subpopulations contain at least deamidated asparagine (N or Asn) residues. For example, asparagine in an asparagine-glycine pair is highly deamidated.
In embodiments, the AAVhu68vp1 nucleic acid sequence has the sequence of SEQ ID NO:7, or a complementary strand thereof, e.g., the corresponding mRNA or tRNA.in certain embodiments, vp2 and/or vp3 protein may additionally or alternatively be expressed from a different nucleic acid sequence other than vp1, e.g., to alter the ratio of vp proteins in the selected expression system in certain embodiments, a nucleic acid sequence encoding the amino acid sequence of AAVhu68 vp3 (about aa203 to about aa 736) of SEQ ID NO:8 that does not have the unique region of vp1 (about 685aa 2 to about aa137) and/or the unique region of vp2 (about 1 to about aa202), or the corresponding mRNA or tRNA (or about nt607 to about nt2211 of SEQ ID NO: 7) of its complementary strand is also provided, in certain embodiments, a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 8542 vp3 (about aa203 to about aa 2211) of SEQ ID NO:8 that does not have the unique region of vp1 (about aa1 to about aa) or about aa 2217, or the corresponding amino acid sequence of SEQ ID NO: 857 or about 22136 or its corresponding strand of SEQ ID NO:8 that does not have the unique region of vp1 (about aa1 to about aa).
However, other nucleic acid sequences encoding the amino acid sequence of SEQ ID NO:8 may be selected for producing the rAAVhu68 capsid, in certain embodiments the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:7, or a sequence having at least 70% to 99% identity to , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to with SEQ ID NO:7 of SEQ ID NO:8 in certain embodiments the nucleic acid sequence has the nucleic acid sequence of about nt412 to about nt2211 of SEQ ID NO:7, or a sequence having at least 70% to 99%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% identity to about nt2211 of SEQ ID NO:7 with vp2 capsid protein (about aa138 to 736) of SEQ ID NO:8 in certain embodiments the nucleic acid sequence has the nucleic acid sequence of about 607 to about 736%, at least about 99%, at least 85%, at least 90%, at least 95%, at least 99% identity to about 2211%, at least 99% identity to about nt of SEQ ID NO:7 with the vp2 capsid protein (about aa138 to 736) of SEQ ID NO: 8.
It is within the skill of the art to design nucleic acid sequences (including DNA (genomic or cDNA) or RNA (e.g., mRNA)) encoding the AAVhu68 capsid, in certain embodiments, the nucleic acid sequence encoding the AAVhu68vp1 capsid protein is provided in SEQ ID No. 7. also see fig. 8B-8 d. in yet other embodiments, a nucleic acid sequence having from 70% to 99.9% identity to SEQ ID No. 7 can be selected to express the
In certain embodiments, asparagine (N) in the N-G pairs in the AAVhu68vp1, vp2, and vp3 proteins is highly deamidated in certain embodiments, the AAVhu68 capsid comprises a subpopulation of AAVvp1, vp2, and/or vp3 capsid proteins that have at least four highly deamidated asparagine (N) positions in the AAVhu68 capsid protein in certain embodiments, about 20% to 50% of the N-N pairs (excluding the N-N triad) exhibit deamidation in certain embodiments, the th N is deamidated in certain embodiments, the second N is deamidated in certain embodiments, deamidation is about 15% to about 25% deamidation in certain embodiments, deamidation is about 259% to about 42% of the protein at positions Q68 vp1, vp2, and vp3, Q8% of the protein for the aahu 68 protein.
In certain embodiments, the rAAVhu68 capsid is further characterized by amidation at D297 of the vp1, vp2 and vp3 proteins. In certain embodiments, based on the numbering of SEQ ID NO:8, about 70% to about 75% amidation in D at position 297 of the vp1, vp2, and/or vp3 proteins in the AAVhu68 capsid.
In certain embodiments, at least Asp of vp1, vp2, and/or vp3 of the capsid isomerizes to D-Asp, these isomers are typically present in an amount of less than about 1% of the Asp at or more of residue positions 97, 107, 384, based on the numbering of SEQ ID No. 8.
In certain embodiments, rAAVhu68 has an AAVhu68 capsid with vp1, vp2, and vp3 proteins having subpopulations comprising a combination of two, three, four, or more deamidation residues at positions listed in the table below. Deamidation in rAAV can be determined using 2D gel electrophoresis and/or mass spectrometry and/or protein modeling techniques. On-line chromatography can be performed using an acclaim pepmap column and a ThermoUltiMate3000RSLC system (Thermo UltiMate3000RSLC) coupled to qxctivehf (Thermo fisherscientific) with a NanoFlex source. MS data were acquired using the data-dependent top-20 method of QOxacteveHF, and the most abundant precursor ions that had not been sequenced were dynamically selected from the survey scan (200-. Sequencing was performed by higher energy collision dissociation fragmentation, where the target value for the 1e5 ion was determined by predictive automatic gain control (predictive auto gain control) and separation of the precursors was performed using a 4m/z window. Survey scans were obtained at a resolution of 120,000 at m/
In certain embodiments, the AAVhu68 capsid is characterized by having at least 45% of the N residues in its capsid protein at least in positions N57, N329, N45 and/or N512 numbered based on the amino acid sequence of SEQ ID NO:8 deamidated in certain embodiments at least about 60%, at least about 70%, at least about 80%, or at least 90% of the N residues at or more of these N-G positions (i.e. N57, N329, N452 and/or N512 numbered based on the amino acid sequence of SEQ ID NO: 8) are deamidated in these and further embodiments, the vhu68 capsid is further characterized by having a protein population in which about 1% to about 20% of the N residues at one or more of the following positions have deamidation in which N residues at least about 1% to about 20% of the N residues are numbered based on the amino acid sequence of SEQ ID NO:8, N94, N253, N304, N270, N477, N45 and/or N512 in certain embodiments comprising a combination of N27, N638, N27 or N27 or N27N.
Optionally, at least Asp of vp1, vp2 and/or vp3 of the capsid isomerizes to D-Asp, optionally, at least T (Thr, serine) of vp1, vp2 and/or vp3 of the capsid is phosphorylated, optionally, at least T (Thr, threonine) of vp1, vp2 and/or vp3 of the capsid is phosphorylated, optionally, at least 2W (trp, tryptophan) of vp2 and/or vp2 of the capsid is oxidized, optionally, at least 2M (Met, M) of vp2, vp2 and/or vp2 of the capsid is oxidized, optionally, at least 2M of vp2, vp2 and/or
In certain embodiments, the AAVhu capsid comprises a heterogeneous population of vp proteins which are the products of a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8, wherein the vp proteins comprise glutamic acid (Glu) at position 67 and/or valine (Val) at position 157, a heterogeneous population of vp proteins which optionally comprise valine (Val) at position 157, and a heterogeneous population of vp proteins, the AAVhu capsid comprises at least one subgroup in which at least 65% of the asparagine (N) in the asparagine-glycine pair at position 57 of the vp protein and at least 70% of the asparagine (N) in the asparagine-glycine pair at position 329, 452 and/or 512 of the vp protein are deamidated based on the residue numbering of the amino acid sequence of SEQ ID NO:8, wherein deamidation results in an amino acid change, wherein deamidation results in a nucleic acid change, as discussed in more detail herein, the deamidation can be at least 99% of asparagine (AA), isoaspartic acid, a tautomedin, or a combination thereof, which encodes at least 99% of the amino acid sequence of the mRNA of at least 99% of the amino acid sequence encoding the amino acid sequence of the protein, or at least 99% of the mRNA encoding the amino acid sequences of the proteins which are complementary SEQ ID protein of at least 99, or at least 99% of the sequences of the proteins, or of the sequences of the polypeptides of the proteins, such as the sequences encoding the sequences of at least 99, or 99, or 99, or 99, or 99, or 99, or 95, 99, 95, or 95, 97, or 95, 97, or 95, 97, or 95, 97, or 95, 97, or 95, 97.
Additionally or alternatively, the rAAVhu capsid comprises at least a subset of vp, vp and/or vp proteins which subpopulation is deamidated at one or more positions N, N113, N252, N253, Q259, N270, N303, N304, N305, N319, N328, N329, N336, N409, N410, N452, N477, N512, N515, N598, Q599, N628, N651, N663, N709, or a combination thereof based on the numbering of SEQ ID NO:8, (e) the rAAV capsid comprises vp, vp and/or vp proteins at one or more positions comprising 1% to 20% deamidation at one or more positions N, N113, N252, N253, Q259, N270, N303, N304, N319, N477, N328, N95% of rAAV proteins based on the numbering of SEQ ID NO:8, or about 10% of rAAV proteins, wherein the subgroup comprising proteins at the ratio of v7% of rAAV proteins comprises the proteins from 1% to 5% of rAAV proteins, 10% or about 10% of rAAV proteins, wherein the subgroup of rAAV proteins comprises the entire vp proteins, the entire v7% of rAAV proteins, 10% of rAAV proteins, the protein, the entire protein, or the protein, rAAV protein, or protein, the protein comprises the protein, or protein, the protein, or the protein, the protein, or the protein, the protein comprises the protein, or the protein, or the protein, or the protein comprises the protein, or the protein, wherein the protein.
In certain embodiments, AAVhu68 is modified to alter glycine in asparagine-glycine pairs to reduce deamidation in other embodiments asparagine is altered to a different amino acid, such as glutamine that deamidates at a slower rate, or to an amino acid lacking an amide group (e.g., glutamine and asparagine contain an amide group), and/or an amino acid lacking an amine group (e.g., lysine, arginine, and histidine contain an amide group), as used herein, an amino acid lacking an amide or amine side group refers to, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, phenylalanine, tyrosine, or tryptophan, and/or proline.
For example, a nucleic acid sequence containing modified AAVhu68vp codons can be generated in which to three codons encoding glycine at positions 58, 330, 453, and/or 513 in SEQ ID NO:8 (arginine-glycine pair) are modified to encode amino acids other than glycine in certain embodiments, a nucleic acid sequence comprising modified arginine codons can be engineered at to three arginine-glycine pairs located at positions 57, 329, 452, and/or 512 in SEQ ID NO:8 such that the modified codons encode amino acids other than arginine in certain embodiments, each modified codon can encode a different amino acid or or more altered codons can encode the same amino acid.
In various embodiments, recombinant adeno-associated virus (rAAV) is provided comprising (A) an AAV capsid comprising one or more of (1) an AAV hu capsid protein comprising an AAVhu vp protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO:8 from 1 to 736, or a vp protein produced by expression of a nucleic acid sequence having at least 70% homology with the nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO:8 from 1 to 736, a Vhu vp protein produced by expression of a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO:8 from at least about amino acid 138 to 736, a vp protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO:8 from at least about amino acid residue 202 to 736, a vhu protein produced by a nucleic acid sequence encoding the predicted amino acid sequence of SEQ ID NO:7 from a nucleotide sequence encoding at least about amino acid residue 412 to 736 of SEQ ID NO:1, or a nucleotide sequence encoding a nucleotide sequence of a nucleotide residue of SEQ ID NO: 2217 which is at least about 70% homologous to 2218, or a nucleotide sequence which is produced by deletion of a nucleotide sequence encoding a nucleotide residue of a vhu V12, a nucleotide residue of a vhu V12, or a nucleotide residue of a nucleotide sequence encoding a nucleotide residue of a nucleotide sequence encoding a nucleotide residue of a nucleotide sequence of a nucleotide residue of a nucleotide sequence of a nucleotide.
In certain embodiments, deamidated glutamine is deamidated to (α) -glutamic acid, gamma-glutamic acid, tautomeric (α) -glutamic acid/gamma-glutamic acid pair, or a combination thereof.
In certain embodiments, the AAVhu capsid comprises a subgroup of proteins having (a) at least 65% of the asparagine (N) in the asparagine-glycine pair at position 57 of the vp protein is deamidated based on the numbering of SEQ ID NO:8, (b) at least 75% of the N in the asparagine-glycine pair at position 329 of the vp, v and vp protein based on the residue numbering of the amino acid sequence of SEQ ID NO:8 is deamidated, (c) at least 50% of the N in the asparagine-glycine pair at position 452 of the vp, v and vp protein based on the residue numbering of the amino acid sequence of SEQ ID NO:8 is deamidated, and/or (d) at least 75% of the N in the asparagine-glycine pair at position 512 of the vp, v and vp protein comprises at least 75% of the proteins in a subgroup of SEQ ID NO: 12, or 92, or from SEQ ID NO: 100, or 200, or from SEQ ID No. 75 to 200, or from SEQ ID No. 7 to 200, or from a subgroup of proteins having at least one or from the amino acid sequence of SEQ ID NO: 100, or from SEQ ID No. 1 to 100, or from SEQ ID No. 7 to 100 to 200, or from a subgroup of proteins having at or from the amino acid sequence of a nucleotide sequence of the amino acid sequence of a protein containing at least one or from SEQ ID NO: 100, or from the amino acid sequence of a protein containing a protein or from SEQ ID NO: 100, or from the polypeptide, or from SEQ ID NO: 100, or from a protein containing at or from the polypeptide, or from the polypeptide or from the nucleotide sequence of the polypeptide or from SEQ ID NO: 100, or from the polypeptide or from SEQ ID NO: 10, or from SEQ ID NO: 12, or from SEQ ID NO: 100 to 10, or from SEQ ID NO: 100 to 10, or from SEQ ID NO:8, or from SEQ ID NO: 92, or from SEQ ID No. 7, or from SEQ ID NO: or from the protein containing at least one or from the protein containing at or from the protein subgroup of the protein containing at or from the protein containing at or the protein containing at least one or from the protein containing at or from the protein containing at least one of the protein containing at or from the protein containing at least one of the protein or from the protein containing at least 100 or from the protein containing at or the protein containing at or from the protein containing at or the protein or from the protein or the protein containing at or the protein containing at least one of the protein containing at or from the protein containing at least one or the protein containing at or more of the protein containing at or from the protein containing at or the protein containing at or from the protein containing at or the protein containing at or from the protein containing at.
In certain embodiments, a composition is provided comprising a mixed population of recombinant adeno-associated virus hu68(rAAVhu68), wherein each rAAVhu68 is independently selected from rAAVhu68 as described herein in certain embodiments, the average AAVhu68 capsid comprises about 60 total capsid proteins in a ratio of about 1 vp1 to about 1 to 1.5 vp2 to 3 to 10 vp3 proteins.
For example, such a production system may comprise (a) an AAVhu68 capsid nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:8, (b) a nucleic acid molecule suitable for packaging into an AAVhu68 capsid, the nucleic acid molecule comprising at least AAV Inverted Terminal Repeats (ITRs) and a non-AAV nucleic acid sequence encoding a gene product operably linked to sequences that direct expression of the product in a host cell, and (c) sufficient AAVrep and helper functions to allow packaging of the nucleic acid molecule into a recombinant AAVhu68 capsid, in certain embodiments the nucleic acid sequence of (a) comprises at least SEQ ID NO:7, or sequences having at least 70% to at least 99% homology with SEQ ID NO:7 encoding the amino acid sequence of SEQ ID NO:8, in certain embodiments the system optionally further comprises a nucleic acid sequence encoding about 203 to about 736 of SEQ ID NO:8 in a human AAVhu 3975, or about 22164 in certain embodiments the human baculovirus system comprising about AAVhup 3975 to about amino acid sequences encoding about SEQ ID NO: 7.
In certain embodiments, a method is provided for reducing deamidation of an AAVhu68 capsid, the method comprising generating an AAVhu68 capsid from a nucleic acid sequence comprising a modified AAVhu68VP codon independently comprising to three arginine-glycine pairs at positions 58, 330, 453 and/or 513 in SEQ ID No. 8 such that the modified codon encodes an amino acid other than arginine in certain embodiments, the method comprising generating an AAVhu68 capsid from a nucleic acid sequence comprising a modified AAVhu68VP codon independently comprising a modified codon in positions 57, 329, 452 and/or to three arginine-glycine pairs at positions 57, 329, 452 and/or 512 in SEQ ID No. 68 5 to three arginine-glycine pairs such that the modified codon encodes an amino acid other than arginine in certain embodiments, each modified codon encodes a different amino acid in certain embodiments, two or more modified codons in certain embodiments, two or more amino acid modifications in the same codon encoding the same amino acid in certain AAVhu 6326, preferably two or more amino acid modifications in the same codon encoding arginine-serine capsid, preferably three arginine-asparagine-glutamine codon pairs in certain embodiments, preferably three arginine-asparagine-serine-amino acid mutations at positions 58, preferably three arginine-serine-asparagine-amino acid mutations at positions 58 in certain embodiments, preferably three amino acid deletion mutants, preferably three amino acid-serine-asparagine-serine-asparagine-serine-amino acid mutations at positions in certain embodiments, preferably three amino acid deletion mutants at positions in certain embodiments, preferably three amino acid deletion mutant AAVhu-asparagine-amino acid pairs in certain embodiments, preferably three different amino acid codon-12, preferably three amino acid pairs in certain embodiments, preferably three amino-asparagine-codon.
As used herein, an "encoded amino acid sequence" refers to an amino acid that is predicted based on translation of a known DNA codon of a reference nucleic acid sequence translated into an amino acid. The following table illustrates the DNA codons and 20 common amino acids, showing the Single Letter Code (SLC) and the three letter code (3 LC).
See, e.g., Gao, et al, proc.natl.acad.sci.u.s.a.100(10),6081-6086(2003) and US2013/0045186a1 other capsids, e.g., those described in WO 2003/042397, WO2005/033321, WO2006/110689, US7588772B2 (which is incorporated herein by reference) can be used in human subjects hi embodiments, the invention provides engineered molecules comprising a spacer sequence between the AAVhu68vp1 coding sequence and the AAVhu68 rep coding sequence.
As noted above, AAVhu sequences and proteins are used to produce rAAV. the following examples describe the production of rAAV vectors with AAVhu or AAV vectors, however, in other embodiments, selection of additional AAV capsids tissue specificity is determined by capsid type, for example, viral vectors with AAVhu are exemplified in the following examples as useful for transducing nasal epithelial cells.sequences of AAVhu are described herein, in addition, methods of producing vectors with AAV capsids and chimeric capsids derived from AAV have been described, see, e.g., US, which is incorporated by reference herein, other AAV serotypes that transduce nasal cells or other suitable targets (e.g., muscle Vp or lung) can be selected as a source of AAV viral vector (dnase resistant viral particle) capsids, including, e.g., AAV, vra, vr6.2, AAV, rh, aah 64R, rh (see, e.g., U.g., U.S. published patent application No. 2007-6760-WO 2009-6760-a), and other AAV serotypes including, AAV sequences that can be used in AAV viral vectors for the production of AAV, or AAV vector, and other AAV vector for use as a replacement for the production of chimeric proteins, or AAV vector for the aforementioned chimeric proteins, which can be found in various AAV vector production of chimeric proteins, or AAV vector, which can be used in various AAV vector-chimeric proteins, as a chimeric gene, or chimeric gene, and in various AAV vector-chimeric gene, including, and in various AAV vector-chimeric gene engineering, or chimeric gene engineering, and in various other AAV vector production of AAV vectors including, AAV vectors, or chimeric gene, as a chimeric gene.
In certain embodiments, AAVhu68 capsids may be useful. For example, such capsids can be used to produce monoclonal antibodies and/or to produce reagents for use in assays for monitoring AAVhu68 concentration levels in gene therapy patients. Techniques for generating useful anti-AAVhu 68 antibodies, labeling such antibodies or empty capsids, and suitable assay formats are known to those skilled in the art.
More typically, the AAVhu68 capsids provided herein are used to produce recombinant AAV vectors in which the engineered nucleic acid sequences are packaged in the AAVhu68 capsid. These recombinant AAV vectors (referred to as "rAAVhu 68" or "rAAVhu 68 vector") and their uses are discussed in more detail elsewhere in this application. These rAAVhu68 vectors are useful for generating recombinant aav (rAAV) vectors that provide good yield and/or packaging efficiency, as well as providing rAAV vectors for transduction of many different cell and tissue types. Such cell and tissue types include, but are not limited to, lung, heart, muscle, liver, pancreas, kidney, brain, hippocampus, motor cortex, cerebellum, nasal epithelial cells, cardiac muscle cells or cardiomyocytes, hepatocytes, pulmonary endothelial cells, muscle cells, pulmonary epithelial cells, islet cells, acinar cells, kidney cells, and/or motor neurons.
In certain embodiments, the vector having the AAVhu68 capsid increases the yield of the packaging vector by at least 15% compared to an AAV 9-based vector. In a comparison between AAVhu68 and AAVrh10, it has been found that AAVhu68 is at a low dose (e.g., about 1x 10) following intraventricular administration9) In a further -step comparison between AAVhu68 and AAV9, it has been found that AAVhu68 is in the cerebellum, motor cortex and hippocampus of the brain following intraventricular administration (e.g., about 1x1011GC) provides better transduction efficiency than AAV9.
A "recombinant AAV" or "rAAV" is a DNase-resistant viral particle comprising two elements, an AAV capsid, and a vector genome comprising at least non-AAV coding sequences packaged within the AAV capsid.
Generally, the term "nuclease resistance" means that the AAV capsid is assembled around an expression cassette, which is designed to deliver genes to host cells and protect these packaged genomic sequences from degradation (digestion) during a nuclease incubation step designed to remove contaminating nucleic acids that may be present in the production process.
In certain embodiments, the non-viral genetic element used to produce the rAAV is referred to as a vector (e.g., a production vector). In certain embodiments, these vectors are plasmids, but the use of other suitable genetic elements is also contemplated. Such a production plasmid may encode sequences that are expressed during rAAV production, e.g., AAV capsid or rep proteins required for production of rAAV, which are not packaged into rAAV. Alternatively, such a production plasmid may carry the vector genome packaged in a rAAV.
As used herein, "vector genome" refers to a nucleic acid sequence packaged within a rAAV capsid that forms a viral particle. Such nucleic acid sequences comprise AAV Inverted Terminal Repeats (ITRs). In this example herein, the vector genome comprises at least 5 'to 3' an AAV5 'ITR, the coding sequence and an AAV 3' ITR. The ITRs from AAV2 (which is different from the AAV from which the capsid is derived) or ITRs other than full-length ITRs may be selected. In certain embodiments, the ITRs are from the same AAV source or a trans-complementary AAV as the AAV that provides rep function during production. In addition, other ITRs may be used. In addition, the vector genome contains regulatory sequences that direct the expression of the gene product. Suitable components of the vector genome are discussed in more detail herein.
Typically, such expression cassettes used to generate viral vectors comprise the hSMN sequences described herein, flanked by packaging signals and other expression control sequences of the viral genome, such as those described herein, e.g., for AAV viral vectors, the packaging signals are 5 'Inverted Terminal Repeats (ITRs) and 3' ITRs.
As used herein, the term "SMN" includes any isoform of SMN that restores a desired function, reduces symptoms, or provides other desired physiological results when the compositions or methods provided herein are delivered. The examples provided herein use the longest isoform, isoform D, which is believed to be the major transcript produced by a gene in a patient unaffected by SMN deficiency or defect. Isoform D provides a protein of 294 amino acids [ see, e.g., NCBI accession no NM _000334/NP _ 000335; ENSEMBL IDENST00000380707], the protein sequence is reproduced in
In certain embodiments, provided herein are engineered human (h) motor neuron Survival (SMN) cDNAs designed to maximize translation compared to the native hSMN sequence (SEQ ID NO: 3). Introns may be introduced upstream of the coding sequence to improve 5' capping and stability of the mRNA. See, e.g., SEQ ID NOS: 15 and 25. These compositions are useful in methods of treating spinal muscular atrophy as described herein. For comparison purposes, an alignment of the native human SMN coding sequence and the engineered cDNA is shown in FIGS. 1B-1C.
The hSMN cDNA sequences described herein can be generated in vitro or synthetically, or by any other suitable method by techniques well known in the art. For example, PCR-based precision synthesis (PAS) of long DNA sequences may be used, as described by Xiong et al, PCR-based cure synthesis of long DNA sequences, Nature protocols1,791-797 (2006). Methods combining the methods of double asymmetric PCR and overlap extension PCR are described by Young and Dong, Two-step total gene synthesis method, Nucleic Acids Res.2004; 32(7) e 59. See also Gordeeva et al, J Microbiol methods, improved PCR-based gene synthesis and its application to the Citrobacter free bacterial gene coding,
In embodiments, rAAVhu68.SMN is a viral vector consisting of an outer component and an inner DNA genome, the outer vector component is an AAVhu68 capsid as defined herein, packaged within the capsid is a single-stranded DNA genome consisting of a human motoneuron survival (hSMN) transgene flanked by two AAV Inverted Terminal Repeats (ITRs), an enhancer, a promoter, an intron, an hSMN1 coding sequence, and a (poly A) signal constituting the hSMN transgene, ITR is a genetic element responsible for genome replication and packaging during vector production, and is a required only viral CIs-element expression of the hSMN coding sequence is driven by a7 promoter, the 7 promoter is a body between the Cytomegalovirus (CMV) immediate early enhancer (C4) and the chicken β actin promoter, the presence of a hybrid between the hCI enhances transcription from the promoter rBG comprising the poly A signal to mediate transcription of human mRNA in the SMN termination scheme, the hSMN1.
In aspects, coding sequences are provided that encode functional SMN proteins, "functional hSMN" refers to a gene encoding an SMN protein that provides a level of biological activity of at least about 50%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of a native motor neuron survivin or a native variant or polymorph that is not associated with a disease.
There are a variety of assays for measuring SMN expression and activity levels in vitro. See, e.g., Tanguy et al, 2015, cited above. The methods described herein may also be combined with any other therapy for treating SMA or symptoms thereof. In certain embodimentsThe standard of care may include norcinasodium (nusnersen), which is FDA and EMA [ SPINRAZATM，Biogen]The SMN2 pre-messenger ribonucleic acid (mRNA) targeted antisense oligonucleotide (ASO) was received see, e.g., U.S. patent nos. 6,166,197, US 6,210,892, US7,101,993, US7,838,657, US 8,110,560, US 8,361,977, US 8,980,853, which is SMN2 directed antisense oligonucleotide administered intrathecally recommended dose is 12mg (5 mL per administration), treatment is started with 4 loading doses, the first 3 loading doses are administered at 14 day intervals, the 4 th loading dose is administered 30 days after the 3 rd dose, the maintenance dose is administrations every 4 months thereafter.
In embodiments, the amino acid sequence of a functional SMN is the amino acid sequence of SEQ ID NO:2 or a sequence sharing 95% homology to with in embodiments, modified hSMN coding sequences are provided, preferably, the modified hSMN coding sequence has less than about 80% homology to , preferably about 75% homology to or less with the full length native hSMN coding sequence (fig. 1B-1C, SEQ ID NO:3) in embodiments, the modified hSMN coding sequence is characterized by an improved translation rate compared to native hSMN after AAV-mediated delivery (e.g., rAAV) in embodiments, the modified hSMN coding sequence shares less than about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 4835% or less with the full length native hSMN coding sequence in 38723 embodiments, the modified hSMN coding sequence shares more than about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 63%, 61%, 4835%, or less with the coding sequence in another embodiment, 95%, 85%, 95%, or more, 95%, or more, 3876%, the coding sequence of the native hSMN coding sequence.
Additionally or alternatively, the compositions or regimens provided herein may be used in combination with treatment using a combination of AAVhu68.SMN stock solutions encoding isoform DSMN proteins and a carrier stock solution encoding a different SMN protein.
In the case of nucleic acid sequences, the terms "percent (%) versus identity", "sequence versus identity", "percent sequence versus identity", or "percent versus identity" refer to residues that are the same in the two sequences when used in corresponding alignment.
In general, when referring to "homo ", "homology" or "similarity" between two different sequences, reference to an "alignment" sequence is to determine "homo ", "homology" or "similarity", an "alignment" sequence 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 as compared to a reference sequence.
Alignment of sequences can be determined by preparing alignments of sequences and by using various algorithms and/or computer programs known or commercially available in the art [ e.g., BLAST, ExPASY; ClustalO; FASTA; using, for example, the Needleman-Wunsch algorithm, the Smith-Waterman algorithm ], alignment is performed using any of a variety of published or commercially available multiple sequence alignment programs.
Examples of such programs include "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME", which are accessible via a Web server on the InternetTM(program in GCG Version 6.1) comparison of polynucleotide sequences. FastaTMAlignment and percent sequence identity which provides the best overlap region between query and search sequences for example Fasta provided in GCG Version 6.1 can be usedTMPercentage of sequence identity between nucleic acid sequences was determined by its default parameters (
Expression cassettes and vectors
In embodiments, the hSMN described herein is genetically engineered into a suitable genetic element (vector) that can be used to generate viral vectors and/or for delivery to a host cell, such as naked DNA, phage, transposons, cosmids, episomes, etc., which deliver the hSMN1 sequences carried thereon.
Thus, in aspects, there are provided adeno-associated viral vectors comprising an AAV capsid and at least expression cassettes, wherein the at least expression cassettes comprise nucleic acid sequences encoding SMN and expression control sequences directing expression of the SMN sequences in a host cell the AAV vector further comprises an AAV ITR sequence in embodiments, ITRs from an AAV different from the AAV providing the capsid, in preferred embodiments, the ITR sequence from AAV2 or a deleted version thereof (Δ ITR) may be conveniently used and expedite regulatory approval, however, ITR from other AAV sources may also be selected where the source of ITR is from AAV2 and the capsid is from another AAV sources, the resulting vector may be referred to as pseudotyped, typically, the AAV vector genome comprises AAV5 ' ITR, hSMN coding sequences and any regulatory sequences, and AAV3 ' ITR, however, other configurations of these elements may be suitable, the full-length versions of which have been described, in which the full-length ITR version is referred to as the full-length ITR, truncated version of AAV3 ' ITR, in which the AAV vector is referred to the full-length ITR, in , embodiments, and the use of the entire length ITR sequence.
In aspects, constructs are provided which are DNA molecules (e.g., plasmids) for generating viral vectors the expression cassettes generally comprise promoter sequences as portions of the expression control sequences, e.g., located between the selected 5' ITR sequence and hSMN coding sequence the illustrative plasmids and vectors described herein use ubiquitous chicken β -actin promoter (CB) and CMV immediate early enhancer (CMV IE) or neuron-specific promoters [ see, e.g., the LockeryLab neuron-specific promoter database, access to the chipook. uoorgeon. edu/promoters. html ]. such neuron-specific promoters include, but are not limited to, e.g., synapsin I(s), (ca/calmodulin-dependent protein kinase II, tubulin α I, neuron-specific enolase and platelet-derived growth factor β chain promoters see, Hioki et al, general Therapy,2007, 14: 11, 2007, 82, which is incorporated by reference herein to the neuron-specific promoters of genes, e.g., the glutamic acid receptor accession nos. 4132, 35, 11, the promoter, the neurotrophin promoter, the Gene promoter
Other promoters, such as constitutive promoters, regulated promoters [ see, e.g., WO2011/126808 and WO2013/04943], or promoters responsive to physiological signals can be used in the vectors described herein, promoters can be selected from various sources, such as the human Cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus promoter (JC polyomavirus promoter), Myelin Basic Protein (MBP) or Glial Fibrillary Acidic Protein (GFAP) promoter, herpes simplex virus (HSV-1) Latency Associated Promoter (LAP), Rous Sarcoma Virus (RSV) Long Terminal Repeat (LTR) promoter, neuron specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, Melanin Concentrating Hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and chicken β -actin promoter.
In addition to the promoter, the expression cassette and/or vector may comprise or more other suitable transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals, sequences which stabilize cytoplasmic mRNA such as WPRE, sequences which enhance translation efficiency (i.e., Kozak consensus sequences), sequences which enhance protein stability, and sequences which enhance secretion of the encoded product, when desired, examples of suitable polyadenylation sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. examples of suitable enhancers are CMV enhancers.
These control sequences are "operably linked" to the hSMN gene sequence. As used herein, the term "operably linked" refers to both the case where the expression control sequence is adjacent to the gene of interest and where the expression control sequence acts in trans or remotely to control expression of the gene of interest.
In embodiments Self-complementary (Self-complementary) AAVs are provided in this context, the abbreviation "sc" refers to Self-complementary "Self-complementary AAV" refers to a construct in which the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double stranded DNA template upon infection, rather than awaiting cell-mediated synthesis of the second strand, the two complementary halves of scAAV will bind to form double stranded DNA (dsdna) units ready for immediate replication and transcription, see, e.g., D M McCarty et al, "Self-complementary adeno-assisted virus (AAV) vector promoter reaction transduction expression of DNAsynthesis", Gene Therapy, (2001 month 8),
Methods of generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art see, for example, U.S. published patent application No.2007/0036760(2007, 2/15), U.S. patent 7790449, U.S. patent 7282199, WO 2003/042397, WO2005/033321, WO2006/110689, and US7588772B2 in systems, transient transfection of producer cell lines with constructs encoding transgenes flanked by ITRs and constructs encoding rep and cap in a second system, transient transfection of packaging cell lines stably supplying rep and cap with constructs encoding transgenes flanked by ITRs in each of these systems, production of viral AAV bodies in response to infection by helper Adenovirus or herpes virus, requiring isolation of contaminating virus, more recently, systems have been developed that do not require helper virus infection for recovery-the helper functions required (i.e. Adenovirus E , E2 , VA and E or UL , UL and UL , and also may be provided by the expression of these promoters by the promoter systems in the context of a transgenic AAV vector production system by the expression of a transgenic AAV vector, e.g. after transient transfection of a Gene expression of a Gene promoter Gene encoding Adenovirus, a Gene encoding AAV, a Gene encoding Human AAV, a transgene, a Gene encoding AAV, a Gene encoding a polypeptide, a polypeptide.
Such other viral vectors may include any virus suitable for gene therapy, including but not limited to adenovirus, herpes virus, lentivirus, retrovirus, bocavirus, suitably produced as a replication-defective viral vector in the context of the production of for these other vectors.
In certain embodiments, engineered aavhu68.SMN vectors are provided, in embodiments, the vector genome of rAAVhu68SMN has the sequence of SEQ ID No. 15 in another embodiments, the vector genome of rAAVhu68SMN has the sequence of SEQ ID No. 25 in certain embodiments, the terms "rAAVhu 68.SMN 1" and "rAAVhu 68. SMN" are used interchangeably.
Compositions and uses
Also provided herein are pharmaceutical compositions. The pharmaceutical compositions described herein are designed for delivery to a subject in need thereof by any suitable route or combination of different routes.
These delivery means are designed to avoid direct systemic delivery of suspensions containing the AAV compositions described herein. Suitably, this may have the benefit of reduced dose, reduced toxicity and/or reduced undesirable immune responses to AAV and/or transgene product compared to systemic administration.
Alternatively, other routes of administration may be selected (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, and other parental routes).
Immunosuppressive co-therapy may be administered for a subject in need thereof, immunosuppressive co-therapy includes, but is not limited to, glucocorticoids, steroids, antimetabolites, T-cell inhibitors, macrolides (e.g., rapamycin or rapalog), and cytostatics, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active against immunophilins, immunosuppressive agents may include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine (mercaptoprine), fluorouracil, actinomycin, anthracyclines (anthracyclines), mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD 3-directed antibodies, anti-IL-2 antibodies, cyclosporines, tacrolimus, sirolimus, IFN- β, IFN- γ, opioids, or TNF- α (tumor necrosis factor-38964) binding agents in certain α embodiments, immunosuppressive therapy may be administered prior to or after gene therapy administration of the same or more doses of a rapamycin, another co-therapy may be administered for a subject in need thereof, or a further dose of two or more (e.g.7, i.7, which may be administered for a dose of one or more than once administration of a dose of a glucocorticoid (e), i.g., may be administered for a dose of a glucocorticoid, which may be administered for a dose of a selected for a dose of a subject in which may be administered for a subject in need of a subject, which may be administered for example, which may be administered for a subject, which may be administered for a dose of a subject, which may be administered for a dose of a subject, which may be.
Raavhun vectors described herein can be administered in a single composition or multiple compositions optionally, two or more different rAAV can be delivered [ see, e.g., WO2011/126808 and WO2013/049493] in another embodiments, such multiple viruses can comprise different replication-deficient viruses (e.g., AAV, adenovirus, and/or lentivirus). alternatively, delivery can be mediated by non-viral constructs, e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA, coupled to various delivery compositions and nanoparticles, including, e.g., micelles, liposomes, cationic lipid-nucleic acid compositions, poly-glycan compositions, and other polymers, lipid and/or cholesterol-based nucleic acid conjugates, and other constructs such as described herein, see, e.g., x.su et al, mol.
In certain embodiments, the raavhu68.sma is purified from any contaminants associated with production prior to storage and/or formulation for delivery to a subject. A variety of suitable purification methods may be selected. Examples of suitable Purification methods are described, for example, International patent application No. PCT/US2016/065970 and its priority documents, filed 2016, 12, 9, 2016, U.S. patent application No. 62/322,071, filed 2016, 4, 13, 4, 2016, and 62/226,357, filed 2015, 12, 11, entitled "Scalable Purification Method for AAV9," which are incorporated herein by reference. For the purification of AAV8, International patent application PCT/US2016/065976 and its priority documents, filed 2016, 12, 9, 2016, U.S. patent application Nos. 62/322,098, 4, 13, 2016, and 62/266,341, filed 2015, 12, 11, and rh10, International patent application No. PCT/US16/66013 and priority documents thereof filed 2016, 12, 9, 2016, U.S. patent application No. 62/322,055 filed 2016, 4, 13, and 62/266,347 entitled "Scalable Purification Method for
For the raavhu68 smn1 vector described herein, quantification of genomic copies ("GC") can be used as a measure of the dose contained in the formulation. any method known in the art can be used to determine the number of Genomic Copies (GC) of the replication-defective viral compositions of the invention methods for performing AAV GC number titration are as follows, a sample of purified AAV vector is first treated with dnase to remove contaminating host DNA from the production process and then the dnase resistant particles are heat treated to release the genome from the capsid, the released genome is then quantified by real-time PCR using primer/probe sets targeting specific regions of the viral genome (e.g. poly a signals). another suitable methods for determining genomic copies are quantitative PCR (qPCR), particularly optimized qPCR or digital droplet PCR [ Lock Martin, et al, Human therapeutics methods 2014ril, 25(2):115-125.doi:10.1089/hgtb.2013.131, published in 20112, 13 th.t. 13, flow cytometry before cell editing, or 3100.
The smavhu 68smn 1 composition can be formulated in dosage units to contain the following amounts of rAAV: about 1.0x109GC to about 9x1015GC (e.g., based on a body weight of about 2.5kg to about 70 kg), including all integers within this rangeOr fractional amount, and preferably 1.0x10 for human patients12GC to 1.0x1014In embodiments, the composition is formulated to contain at least 1x10 per dose9、2x109、3x109、4x109、5x109、6x109、7x109、8x109Or 9x109In another embodiments, the composition is formulated to contain at least 1x10 per dose10、2x1010、3x1010、4x1010、5x1010、6x1010、7x1010、8x1010Or 9x1010In another embodiments, the composition is formulated to contain at least 1x10 per dose11、2x1011、3x1011、4x1011、5x1011、6x1011、7x1011、8x1011Or 9x1011In another embodiments, the composition is formulated to contain at least 1x10 per dose12、2x1012、3x1012、4x1012、5x1012、6x1012、7x1012、8x1012Or 9x1012In another embodiments, the composition is formulated to contain at least 1x10 per dose13、2x1013、3x1013、4x1013、5x1013、6x1013、7x1013、8x1013Or 9x1013In another embodiments, the composition is formulated to contain at least 1x10 per dose14、2x1014、3x1014、4x1014、5x1014、6x1014、7x1014、8x1014Or 9x1014In another embodiments, the composition is formulated to contain at least 1x10 per dose15、2x1015、3x1015、4x1015、5x1015、6x1015、7x1015、8x1015Or 9x1015In embodiments, for human administration, the dosage can be 1x10 per dose10To about 1x1015GC, including all whole or fractional amounts within the range.
In certain embodiments, the dose may be about 1x109GC/g brain Mass to about 1x1012GC per g brain mass. In certain embodiments, the dose may be about 3x1010GC/g brain mass to about 3x1011GC per g brain mass. In certain embodiments, the dose may be about 5x1010GC/g brain mass to about 1.85x1011GC per g brain mass.
In embodiments, rAAVhu68.SMN1 can be at least about 1x109GC to about 1x1015Or about 1x1011To 5x1013For example, a volume of about 1 μ L to 150mL may be selected, a higher volume may be selected for adults, generally, a suitable volume is about 0.5mL to about 10mL for newborns, a volume of about 0.5mL to about 15mL may be selected for infants, a volume of about 0.5mL to about 20mL may be selected for toddlers, a volume of up to about 30mL may be selected for children, a volume of up to about 50mL may be selected for adolescents and juveniles, in yet other embodiments, the patient may receive intrathecal administration, a volume of about 5mL to about 15mL, or about 7.5mL to about 10mL other suitable volumes and doses may be determined.
Delivery of the above raavhu68.smn1 to a host cell can be performed according to published methods. In certain embodiments, for administration to a human patient, the rAAV is suitably suspended in an aqueous solution comprising saline, a surfactant, and a physiologically compatible salt or mixture of salts. Suitably, the formulation is adjusted to a physiologically acceptable pH, for
In embodiments, a bifunctional block copolymer surfactant terminating in a primary hydroxyl group is selected, e.g.
In examples, the formulation can comprise, for example, a buffered saline solution comprising sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfate 7H) in water2O), potassium chloride, calcium chloride (e.g., CaCl. multidot.2H)2Suitably, for intrathecal delivery, the osmolarity is in a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine. mediscape. com/-article/2093316-overview. optionally, for intrathecal delivery, commercially available diluents can be usedAs suspending agents, or in combination with another suspending agents and other optional excipients see, e.g., Elliots
sodium chloride, USP 73mg
Sodium bicarbonate, USP 19mg
Dextrose, USP 8mg
Magnesium sulfate 7H2O, USP 3mg
Potassium chloride, USP 3mg
Calcium chloride 2H2O, USP 2mg
Disodium phosphate 7H2O, USP 2mg
Water for injection, USP make-up to 10mL
Electrolyte concentration:
the molecular formula and molecular weight of the component are:
composition (I)
Molecular formula
Molecular weight
Sodium chloride
NaCl
58.44
Sodium bicarbonate
NaHCO3
84.01
Dextrose
C6H12O6
180.16
Magnesium sulfate 7H2O
Mg2SO4·7H2O
246.48
Potassium chloride
KCl
74.55
Calcium chloride 2H2O
CaCl2·2H2O
147.01
Disodium phosphate 7H2O
Na2HPO4·7H2O
268.07
The pH of the Elliots B solution was 6 to 7.5 and the osmolality was 288mOsmol per liter (calculated). In certain embodiments, the composition comprising the raavhu68.smn1 gene is delivered at a pH range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8. For intrathecal delivery, a pH of greater than 7.5 may be required, for example 7.5 to 8, or 7.8.
Such formulations may comprise a buffered saline solution comprising sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and mixtures thereof in water, e.g., Harvard's buffer solution
In another embodiments, the formulation can contain a buffered saline solution comprising 1mM sodium phosphate (Na)3PO4) 150mM sodium chloride (NaCl), 3mM potassium chloride (KCl), 1.4mM calcium chloride (CaCl)2) 0.8mM magnesium chloride (MgCl)2) And 0.001%
Cerebrospinal fluid (CSF) composition
In still other embodiments, the formulation may contain or more penetration enhancers examples of suitable penetration enhancers may include, for example, mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, or EDTA.
In another embodiments, the composition comprises a carrier, diluent, excipient, and/or adjuvant one of skill in the art can readily select an appropriate carrier in view of the indication for which the transferred virus is intended, for example, suitable carriers include saline, which can be formulated with various buffer solutions (e.g., phosphate buffered saline).
Optionally, the compositions of the invention may contain, in addition to the rAAV and the carrier, other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.
Suitably, the compositions described herein comprise an effective amount of or more AAVs suspended in a pharmaceutically suitable carrier and/or admixed with a suitable excipient designed for delivery to a subject by injection, osmotic pump, intrathecal catheter, or for delivery by other means or routes, hi examples, the compositions are formulated for intrathecal delivery hi embodiments intrathecal delivery comprises injection into a spinal canal, such as the subarachnoid space.
The viral vectors described herein can be used in the preparation of a medicament for delivering hSMN to a subject in need thereof (e.g., a human patient), providing functional SMN to a subject, and/or for treating spinal muscular atrophy. The course of treatment may optionally include repeated administration of the same viral vector (e.g., AAVhu68 vector) or a different viral vector (e.g., AAVhu68 and AAVrh 10). Other combinations of the viral vectors and non-viral delivery systems described herein may alternatively be used.
As used herein, the term "intrathecal delivery" or "intrathecal administration" refers to a route of drug administration by injection into the spinal canal, more particularly into the subarachnoid space, so that it reaches the cerebrospinal fluid (CSF). intrathecal delivery may include lumbar puncture, intraventricular (including Intracerebroventricular (ICV)), subarachnoid/intracisternal, and/or C1-2 puncture.
As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to the route of administration of a drug directly into the cerebrospinal fluid of the cisbellomulum cerebellomulum, more specifically by means of an occipital puncture (subperiocitrial puncture) or by direct injection into the cisbellomulum or by means of a permanently located tube.
In embodiments, the delivery can be performed using the devices described herein.
Devices and methods for delivering pharmaceutical compositions into the cerebrospinal fluid
In aspects, the carrier provided herein can be administered intrathecally by the method and/or device provided in this section and described in embodiments and step in FIG. 7 alternatively other devices and methods can be selected the method comprising the steps of advancing a spinal needle into the cisterna magna of the patient, connecting segments of flexible tubing to the proximal hub of the spinal needle and connecting the output port of the valve to the proximal end of the flexible tubing, after said advancing and connecting steps and after allowing the tubing to self-fill by cerebrospinal fluid (self-prime) of the patient, connecting th container containing doses of isotonic solution to the flush inlet of the valve, then connecting a second container containing doses of the pharmaceutical composition to the carrier inlet of the valve, after connecting the th and second containers to the valve, opening the fluid flow path between the carrier inlet and the outlet of the valve and injecting the pharmaceutical composition into the patient through the spinal needle, and after injecting the pharmaceutical composition, opening the fluid flow path through the flush inlet and the outlet of the valve and injecting the pharmaceutical composition into the patient to flush the spinal needle.
In another aspect , a device for intracisternal delivery of a pharmaceutical composition is provided that includes a th container containing doses of the pharmaceutical composition, a second container containing an isotonic solution, and a spinal needle through which the pharmaceutical composition can be expelled from the device directly into cerebrospinal fluid within the cisterna magna of a patient, the device further including a valve having a th inlet interconnected with the th container, a second inlet interconnected with the second container, an outlet interconnected with the spinal needle, and a luer lock (luer lock) for controlling flow of the pharmaceutical composition and the isotonic solution through the spinal needle.
As used herein, the term Computed Tomography (CT) refers to radiography, in which a three-dimensional image of a body structure is computer-constructed from an series of planar cross-sectional images taken along an axis.
The apparatus or
For example, each
Likewise,
Alternatively,
In the illustrated embodiment, valve 16 is provided as a four-way stopcock (stopcock) having a rotating
In the embodiment shown, the valve 16 is connected to the end of lengths of
In the illustrated embodiment, the
In use, the
The above described assembly is allowed to be "self-filled" by the patient's CSF, then a pre-filled saline
After injection of the contents of
After flushing the assembled device with saline, the entire assembled device (including the needle, extension tube, stopcock, and syringe) is slowly removed from the subject and placed on a surgical tray for disposal into a biohazard waste container or rigid container (for the needle).
Screening processes that may ultimately lead to Intracisternal (IC) procedures can be conducted by the primary investigator. The primary investigator can describe the procedure, the administration procedure itself, and all potential safety risks in order to make the subject (or designated caregiver) fully informed. Medical history, concomitant medications, physical examination, vital signs, Electrocardiogram (ECG), and laboratory test results are obtained or performed and provided to neuroradiologists, neurosurgeons, and anesthesiologists for screening assessment of subject eligibility for IC procedures.
For example, on "
In addition, head/neck MRA/MRV may be obtained according to an institutional protocol (i.e., subjects with internal/epidural surgery may be excluded or may require further tests (e.g., radionucleotide encephalography)) to allow for adequate assessment of CSF flow and identification of possible blockage or lack of communication between CSF spaces.
Neuroradiologists, neurosurgeons, and anesthesiologists ultimately discuss and determine whether each subject qualifies for the IC procedure based on all available information (scans, medical history, physical examinations, laboratories, etc.). Pre-operative assessments can also be obtained from "day-28" to "
Prior to the IC procedure, the CT Suite (CT Suite) will be confirmed to have the following equipment and drugs: adult Lumbar Puncture (LP) kits (provided by the institution); BD (Becton Dickinson)22 or 25 gauge x3-7 "spinal needle (Quincke bevel); a coaxial guiding needle, used at the discretion of the interventionalist (for introducing spinal needles); a 4-way small-hole plug valve with a rotary (Spin) Gonluer lock; a T-connector extension (tube) with female luer lock adapter, about 6.7 inches in length; omnipaque180 (iohexol) for intrathecal administration; iodinated contrast agents for Intravenous (IV) administration; injection of 1% lidocaine solution (if not provided in the adult LP kit); pre-filled 10cc saline (sterile) flush syringe; a radiopaque marker; surgical preparation equipment/shaver; a pillow/support to allow for proper positioning of the intubated subject; tracheal intubation equipment, general anesthesia machines and mechanical ventilators; an intraoperative neurophysiological monitoring (IONM) device (and personnel required); and a 10cc syringe containing the carrier; prepared and shipped to the CT/Operating Room (OR) suite according to a separate pharmacy manual.
Informed consent for the procedure was confirmed and recorded in medical records and/or research documentation. Separate consent was obtained from radiology and anesthesiology staff for this procedure, as required by the institution. According to institutional guidelines (e.g., two IV access sites), the subject may conduct venous access within the appropriate hospital care unit. Intravenous infusion is at the discretion of the anesthesiologist. The subject may be induced and subjected to endotracheal intubation and general anesthesia in an appropriate patient care unit, holding area, or surgical/CT procedure kit, according to the judgment of the anesthesiologist and institutional guidelines.
A lumbar puncture was performed by first removing 5cc of cerebrospinal fluid (CSF) and then injecting contrast medium intrathecally (Omnipaque 180) to help visualize the cisterna magna. Appropriate subject positioning manipulations may be performed to facilitate the diffusion of contrast agent into the cisterna magna.
An Intraoperative neurophysiological monitoring (IONM) device is attached to the subject. The subject was placed on a CT scanner table in a prone or lateral position. There must be enough staff to ensure the safety of the subject during transport and positioning. If deemed appropriate, the subject may be positioned in such a way that: neck flexion is provided to an extent determined to be safe during pre-operative assessment, and normal neurophysiological monitoring signals are recorded after positioning.
The presence and designation of the following staff in the field can be confirmed: an interventionalist/neurosurgeon performing the procedure; anesthesiologists and respiratory technicians; nurse and physician assistants; CT (OR) technicians; a neurophysiologist technician; and a field coordinator. The "time-out" can be done according to a joint committee/hospital protocol to verify that all necessary equipment is present in the correct subject, procedure, site, location, and room. The primary site investigator may then confirm with the staff that he/she can proceed to prepare the subject.
The subjacent skin of the subject was shaved as needed. If deemed necessary by the interventionalist, a CT scout image is performed, followed by CT with preoperative planning of IV imaging to locate the target location and image the vasculature. After the target site (cisterna magna) and planned needle trajectory are determined, the skin is prepared and covered using sterile techniques according to institutional guidelines. Radiopaque markers are placed on the target skin location according to the instructions of the interventionalist. The skin under the marker was anesthetized by infiltration with 1% lidocaine. The 22G or 25G spinal needle is then advanced into the cisterna magna, optionally using a coaxial guide needle.
After needle advancement, CT images (ideally ≦ 2.5mm) were acquired using a mechanical device using the thinnest CT slice thickness possible. Continuous CT images are obtained using the lowest radiation dose possible that allows for adequate visualization of the needle and associated soft tissue (e.g., paraspinal muscles, bone, brainstem, and spinal cord). Proper needle placement is confirmed by observing the CSF in the needle hub and visualization of the needle tip within the cerebellum.
The interventionalist confirms that the carrier syringe is located near, but outside of, the sterile field. Gloves, face masks and eye protection devices are worn by personnel of the ancillary procedures within the sterile field prior to handling or administering the pharmaceutical composition in the carrier syringe.
The extension tubing was attached to the inserted spinal needle, which was then attached to the 4-way stopcock, once the device was "self-filled" by the subject's CSF, a 10cc pre-filled saline flush syringe was attached to the flush inlet of the 4-way stopcock, and the carrier syringe was then provided to the interventionalist and attached to the carrier inlet on the 4-way stopcock.
After the contents of the carrier syringe are injected, the rotational lock of the stopcock valve is rotated to the second position so that the stopcock valve and needle assembly can be flushed with 1-2cc of saline using the attached prefilled flush syringe.
Upon readiness, the intervener then notifies the staff to remove the device from the subject. In a single motion, the needle, extension tube, stopcock, and syringe are slowly removed from the subject and placed on a surgical tray for disposal into a biohazard waste container or hard container (needle).
The needle insertion site was examined for signs of bleeding or CSF leakage and treated as instructed by the investigator. The site is bandaged with gauze, surgical tape, and/or Tegaderm dressing as indicated. The subject was then removed from the CT scanner and placed on a stretcher. There are enough staff to ensure the safety of the subject during transport and positioning.
Anesthesia was stopped and the subject was cared for following institutional guidelines for post-anesthesia care. The neurophysiological monitor is removed from the subject. During recovery, the subject should be slightly elevated (-30 degrees) on the stretcher head. Subjects were transported to the appropriate post-anesthesia care unit according to institutional guidelines. After the subject has sufficiently regained consciousness and the condition has stabilized, he is allowed to enter the appropriate floor/ward for protocol-specified evaluation. Neurological assessments will be performed according to protocols with the primary investigator supervising subject care in cooperation with hospitals and researchers.
In embodiments, a method for delivering a composition provided herein includes the steps of advancing a spinal needle into a cisterna magna of a patient, connecting segments of flexible tubing to a proximal hub of the spinal needle and connecting an output port of a valve to a proximal end of the flexible tubing, after said advancing and connecting steps and after allowing the tubing to self-fill by cerebrospinal fluid of the patient, connecting a container containing doses of an isotonic solution to a flushing inlet of the valve, then connecting a second container containing doses of a pharmaceutical composition to a carrier inlet of the valve, after connecting said and second containers to the valve, opening a fluid flow path between the carrier inlet and outlet of the valve and injecting the pharmaceutical composition through the spinal needle, after injecting the pharmaceutical composition, opening the fluid flow path through the flushing inlet and outlet of the valve and injecting the isotonic solution into the spinal needle to flush the pharmaceutical composition into the patient.
In the above method, the valve may be a stopcock valve having a rotary luer lock adapted to rotate to a position allowing flow from the carrier inlet to the outlet while preventing flow through the irrigation inlet, and to a second position allowing flow from the irrigation inlet to the outlet while preventing flow through the carrier inlet, and wherein the rotary luer lock is located at the position when the pharmaceutical composition is injected into a patient and is located at the second position when the pharmaceutical composition is irrigated into the patient by an isotonic solution.
In certain aspects, the method uses a device constructed of at least containers for holding doses of a pharmaceutical composition, a second container containing an isotonic solution, a spinal needle through which the pharmaceutical composition can be expelled directly from the device into cerebrospinal fluid within the cisterna magna of the patient, and a valve having a inlet interconnected with the container, a second inlet interconnected with the second container, an outlet interconnected with the spinal needle, and a luer lock for controlling the flow of the pharmaceutical composition and the isotonic solution through the spinal needle, hi certain embodiments, the valve is a stopcock having a rotary luer lock adapted to rotate to a position, allowing flow from the inlet to the outlet while preventing flow through the second inlet, and to a second position allowing flow from the second inlet to the outlet while preventing flow through the inlet, optionally, the valve is a four-way stopcock having a rotary luer lock, hi certain embodiments, the second container and the second container are containers and the spinal needle are connected to a spinal needle in certain embodiments, the spinal needle may be a flexible introducer needle connected to the spinal needle 3618, in certain embodiments, the spinal needle may be connected to a spinal needle in a flexible tubing 3618, a spinal needle, in certain embodiments, a flexible tubing interconnecting section of the spinal needle.
The method and the device can each optionally be used for intrathecal delivery of the compositions provided herein. Alternatively, other methods and devices may be used for such intrathecal delivery.
In embodiments, a single dose of aavhu68.SMA provided herein is administered to an adult (at least 18 years old (≧ 18)) with a genetically confirmed clinical history of 5q SMA and/or
In embodiments, a syringe containing an appropriate concentration of carrier is used, before carrier administration, a lumbar puncture is made to remove a predetermined volume of CSF, and then iodinated contrast media (IC) is injected intrathecally to aid in visualization of the relevant anatomy of the cisterna magna.an Intravenous (IV) contrast media may be administered as a substitute for intrathecal contrast media either before or during needle insertion.the decision to use IV or IC contrast media is decided by the interventionalist.the patient is anesthetized, intubated and positioned on the operating table.an intraoperative neurophysiology monitoring (IONM) device is attached to the participant.an injection site is prepared and covered using sterile techniques.under fluoroscopic guidance, a spinal needle (22-25G) is advanced into the cisterna magna larger introducer needle is used to assist needle placement.after needle placement is confirmed, an extension device is connected to the spinal needle and allowed to fill the patient.at the discretion of the interventionalist, a syringe containing contrast material may be connected to the extension device and a small amount is injected to confirm that the placement of the needle in the cisterna.5. the injection of the contrast media is slowly delivered from the syringe, a syringe containing a volume of the
As used herein, a "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, e.g., a monkey, chimpanzee, baboon, or gorilla in embodiments, the subject is a human patient having SMA.
The international SMA alliance classification defines multiple degrees of severity of SMA phenotype based on age of onset and motor development milestones. SMA0 is named to reflect prenatal episodes and severe joint contractures, facial diploplegia, and respiratory failure. SMA type I (wedneshi-hofmann disease I) is the most severe postpartum form, with onset within 6 months after birth. The patient is unable to sit up and has severe respiratory function disorders. Type II SMA is an intermediate form that is diseased within the first 2 years; the child can sit up but cannot walk. The clinical course is variable. Type III (also known as kugelberg-welan disease) begins after 2 years of age and often has a chronic progression. The child may stand and walk independently, at least during infancy. Adult form (type IV) is the mildest, with onset after 30 years of age; the reported cases are rare and their prevalence is not accurately known.
In certain instances, SMA is detected in a fetus of about 30 to 36 weeks of pregnancy in which case it may be desirable to treat the neonate as soon as possible after delivery, it may also be desirable to treat the fetus in utero. accordingly, there are provided methods of rescuing and/or treating a neonatal subject suffering from SMA comprising the step of delivering the
In embodiments, neonatal treatment is defined as administration of the hSMN construct as described herein within 8 hours, the first 12 hours, the first 24 hours, or the first 48 hours of delivery hi another embodiments, particularly for primates (human or non-human), neonatal delivery is within a period of about 12 hours to about 1 week, 2 weeks, 3 weeks, or about 1 month, or after about 24 hours to about 48 hours hi another embodiments, the composition is delivered after onset of symptoms for tardive SMA, in embodiments, treatment of the patient (e.g., injections) begins before years of life, in certain embodiments, e.g., for infants, the construct is re-administered after, e.g., 1 year of age, optionally, more than re-administrations are allowed, such re-administration can use the same type of vector, a different viral vector, or by the non-viral delivery described herein, in another embodiment, after , after 352 years of age, or after 365 years of age, or after 3611 years of age.
Hsmn is delivered as described herein to achieve a desired result, i.e., to treat SMA or symptoms thereof other desired results include reducing muscle weakness, increasing muscle strength and tone, preventing or reducing scoliosis, or maintaining or increasing respiratory health, or reducing tremors or tics.
In certain embodiments, the therapeutic efficacy of a composition containing aav.hsmn provided herein can be assessed by or more of the following parameters such a score can be at 52 weeks, or longer or shorter intervals, e.g., 8 weeks, 12 weeks, 36 weeks, 48 weeks, or times therebetween, comparison of the RULM score to a baseline score at predetermined times after administration, measure motor function by a 6MWT test or 10 meter walking time in ambulatory subjects, measure motor function by a 9-hole spike test (ambulatory and non-ambulatory) and a 4-step climbing test (ambulatory only), measure lung function by Forced Vital Capacity (FVC), maximum pressure (MEP), Maximum Inspiratory Pressure (MIP), change in ulnar and fibular CMAP amplitudes compared to baseline.
As used herein, 6MWT is a measure of the distance covered by an ambulatory subject in 6 MWT. The 6MWT is performed in a long low flow corridor located indoors. A distance of 30 meters will be marked with orange cones at both ends. The start thread is marked with a brightly colored tape.
RULM modified Upper limbs Module consisting of 20 motor tasks performed by upper limbs selected by the subject the assessor rated the performance of each task as 0-2.
9-hole nail test 9-HPT is a simple, standardized, quantitative test of upper limb function both dominant (dominant hand) and non-dominant (non-dominant hand) tests twice, the subject sits on a table with a small shallow container containing nine nails and a wood or plastic block containing nine empty holes, upon a start command when starting the stopwatch, the subject times picks up the nine nails, places them in the nine holes, and once they enter the holes, then times remove them as soon as possible, replace them in the shallow container, record the total time to complete the task, two consecutive trials with the dominant hand, then immediately two consecutive trials with the non-dominant hand, the score for 9-HPT is the average of the four trials, average the two trials per hand, convert to the inverse of the average time per hand, and then average the two inversions.
10m walking time: the 10 meter walking time is a measure of the time required to walk 10 meters. The test will be performed in a long low flow straight corridor located indoors. A distance of 10 meters will be marked with orange cones at both ends. The start thread is marked with a brightly colored tape.
4, climbing in a ladder way: the 4-step climbing test assesses the time required for a subject to ascend and descend 4 steps. The task will be performed by a trained evaluator, which will ensure that the subject is able to safely complete the task. The steps must have a height of 16-20 cm and handrails. Subjects were instructed to ascend and descend as quickly as possible in a safe manner. The time required to complete the task and the need to use the handrail are recorded.
Electrophysiological studies were performed to assess the function of the locomotor unit. CMAP: motor nerve conduction studies of the ulnar nerve are preferentially performed on the right arm unless there is convincing reason to avoid studying the limb (e.g., pre-disease superimposed nerve injury). This involves supplying current to the nerve and recording motor responses in the muscle. This response is called compound muscle action potential (compound muscle action potential). The height and area (amplitude and AUC) of the CMAP can be measured.
Functional and fatigue rating scale:
the PedsQL fatigue Scale, an adult report, is the PedsQL multidimensional fatigue Scale (PedsQL MultidimensionationaFatigue Scale) is questionnaires that assess -like fatigue (6), sleep and rest (6), and cognitive fatigue (6).
SMA-FRS is an easily performed ordinal rating scale (scoring scale) based on 10 aspects of daily life activities, each subset was scored from 0 (fully dependent) to 5 (fully independent) by the subject or caregiver, with a maximum score of 50.
In embodiments, the order of assessment is performed for the patient in the following order PedsQL (fatigue Scale), SMA-FRS (functional rating Scale), 6MWT, 15 minutes rest (minimal), RULM, 9-hole nail test, 10 meters walk, 15 minutes rest (minimal), 4-step climb, PFT, and ulnar and fibular CMAP.
Prior to treatment, SMA patients can be evaluated for neutralizing antibodies (Nab) to the capsid of the rAAV vector used to deliver the hSMN-1 gene. Such nabs may interfere with transduction efficiency and reduce therapeutic efficacy. SMA patients with baseline serum Nab titers of ≦ 1:5 to ≦ 1:20, or ≦ 1:2.5 to ≦ 1:10 are good candidates for treatment with the rAAV.hSMN1 gene therapy regimen. Treatment of SMA patients with serum Nab titers >1:5 may require combination therapy, e.g., transient co-treatment with an immunosuppressive agent prior to and/or during treatment for raav. Optionally, immunosuppressive co-therapy can be used as a prophylactic measure without prior assessment of neutralizing antibodies to the AAV vector capsids and/or other components of the formulation. In certain embodiments, advanced immunosuppressive therapy may be desired to prevent potential adverse immune responses to hSMN transgene products, particularly in patients with little levels of SMN activity, where the transgene product may be considered "foreign".
Immunosuppressant therapies for such co-therapies can include, but are not limited to, glucocorticoids, steroids, antimetabolites, T cell inhibitors, macrolides (e.g., rapamycin or rapalog), and cytostatics, including alkylating agents, antimetabolites, cytotoxic antibiotics, antibodies, or agents active on immunophilins.immunosuppressant inhibitors can include nitrogen mustards, nitrosoureas, platinum compounds, methotrexate, azathioprine, mercaptopurine, fluorouracil, actinomycins, anthracyclines, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD 3-directed antibodies, anti-IL-2 antibodies, cyclosporines, tacrolimus, sirolimus, IFN- β, IFN- γ, opioids, or TNF- α (tumor necrosis factor- α) binding agents in certain embodiments, immunosuppressive therapies can be initiated prior to gene therapy administration.such therapies can include co-administration of two or more drugs (e.g., prednisone, mycophenolate (MMF) and/or rapamycin) on the same day day, i.e.g., gene therapy can be continued on the same day, on the same day as administration of these drugs, on the same day of about 1, or on a more day of about 30, on the same day of administration of these days, on the same day of about 1, on the same day of another day of these drugs, on the same day of about 30 days of theseIn another embodiments, the patient receives continuous treatment of norcisartan sodium and monitors a reduction or elimination of the need for such norcisartan sodium treatment after gene therapy the patient receiving raavhu68.sma may receive other therapies including, but not limited to, pyridostigmine [ UMC Ultrecht [ ]]、RO7034067[Hoffman-LaRoche]Celecoxib, CK-2127107[ Astellas Pharma ]]. In certain embodiments, the efficacy of raavhu68.sma is measured by a reduction in the frequency and/or dose of such co-therapy. In certain embodiments, since AAVhu68-SMN1 therapy, while durable, may not result in high correction desired by the selected patient, conversion to norcisazona sodium (Spinraza) may be required as early as 6 months after AAVhu68-SMN1 treatmentTM) Therapy comprising administering to a patient nocifenesin sodium (Spinraza)TM) Can be considered co-administration of the two agents. See also Wang et al, Consensuss State for Standard of Care in mineral muscle Atcopy, which provides a discussion of the current Standard of Care for SMA, and www.ncbi.nlm.nih.gov/books/NBK 1352/.
For example, when nutrition in SMA is of concern, placement of the gastrostomy tube is appropriate. As respiratory function deteriorates, tracheotomy or non-invasive respiratory support is provided. Sleep disordered breathing may be treated by nighttime use of continuous positive airway pressure. If the forced vital capacity is more than 30-40%, the scoliosis operation of the SMA II and SMA III patients can be safely carried out. Powered chairs and other devices may improve quality of life. See also U.S. patent No. 8211631, which is incorporated herein by reference.
It should be noted that the terms " (a)" or " (an)" refer to or more accordingly, the terms " or more" and "at least " are used interchangeably herein without numerical limitation.
The words "comprise", "comprising" and "contain" are to be interpreted inclusively rather than exclusively. The word "consisting of and variants thereof should be interpreted exclusively rather than inclusively. Although various embodiments in the specification have been presented using the language "comprising," in other instances related embodiments are also intended to be interpreted and described using the language "consisting of or" consisting essentially of.
The term "about" as used herein, unless otherwise specified, means 10% (± 10%) different from the stated reference value.
As used herein, "disease," "disorder," and "condition" are used interchangeably to indicate an abnormal state in a subject.
The term "expression" is used herein in its broadest sense of and includes the production of RNA or RNA and protein.
The term "translation" relates to a process at the ribosome where the mRNA chain controls the assembly of amino acid sequences to produce a protein or peptide.
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, by reference to the disclosure, provide those skilled in the art with -general guidance for the terms used in this application.
The following examples are illustrative only and are not intended to limit the present invention.
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