Injection of single stranded adeno-associated virus 9 or self-complementary adeno associated virus 9 into cerebrospinal fluid

Abstract

To provide compositions and methods for targeting the treatment to the central nervous system.SOLUTION: Disclosed is a method for delivering a non-secreted therapeutic protein to the central nervous system of a human subject in need thereof, the method comprising a step of administering an effective amount of self-complementary adeno-associated virus serotype 9 (scAAV9) encoding a non-secretive therapeutic protein, where the scAAV9 is administered by injection to the cerebrospinal fluid via the intracerebroventricular or intrathecal cisternal or intrathecal lumbar route of administration.SELECTED DRAWING: None

Description

[Technical field]

[0001] region Compositions and methods for targeting treatment to the central nervous system (CNS); specifically, dosing regimens utilizing adeno-associated viruses (AAV), specifically AAV9, administered by injection into the cerebrospinal fluid (CSF) via intracerebroventricular (ICV) or intrathecal (cisternal or lumbar) routes, to target treatment to different cells within the CNS are described.

[0002] Parties to the Joint Research Agreement Emory University and Regenxbio are parties to a collaborative research agreement. [Background technology]

[0003] background Gene therapy has long been studied in animal models to treat diseases of the central nervous system (CNS). A variety of inherited metabolic diseases, such as lysosomal storage diseases, affect the brain, causing mental retardation and neurodegeneration. CNS disorders substantially affect the quality of life of patients and their families, resulting in loss of independence and communication difficulties. Direct delivery of therapeutic genes to the brain offers a potential treatment for these diseases that may avoid the problems found with systemic treatment. For example, neutralizing antibodies in the CNS are of less concern (Gray et al., 2013, Gene Therapy 20:450-459), and although the risk of immune response to vectors and / or transgenes appears to be relatively low (Mingozzi and High, Blood. 2013 Jul 4;122(1):23-36), this risk is not yet zero (Ellinwood et al., Mol Ther. 2011 Feb;19(2):251-9).

[0004] To treat disorders that affect cells throughout the CNS, such as lysosomal storage diseases, one challenge for gene therapy is to achieve adequate transduction throughout the brain and spinal cord. One vector, adeno-associated virus serotype 9 (AAV9), has the ability to transduce cells of the CNS and demonstrates the ability to bypass the blood-brain barrier (BBB). Following intravenous injection of high doses of AAV9 vectors, studies have demonstrated transduction of the CNS in rodents and non-human primates (NHPs) (e.g., Foust, et al., 2009, Nat. Biotechnol. 27:59-65 (Non-Patent Document 4); Duque et al., 2009, Mol. Ther. 17:1187-1196 (Non-Patent Document 5); Fu et al., 2011, Mol. Ther, 19:1025-1033 (Non-Patent Document 6); and Gray et al., 2011, Mol. Ther. 19:1058-1069 (Non-Patent Document 7)). Spinal cord distribution of AAV9 following intrathecal administration to neonatal mice, pigs, and monkeys has also been investigated (Passini et al., 2014, Human Gene Therapy 25:619-630). A recent study compared vascular delivery of AAV9 to CSF ​​delivery in NHPs. Both delivery routes were reported to produce similar distribution patterns and tropism (astrocyte tropism greater than neuronal tropism). CSF administration produced greater transduction of the CNS but did not shield against AAV antibodies (Samaranch et al., 2012, Hum Gene Ther 23:382-389). It is anticipated that substantial innovation will be required to address the significant challenges raised in these and other studies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Gray et al., 2013, Gene Therapy 20:450-459 [Non-Patent Document 2] Mingozzi and High, Blood. 2013 Jul 4;122(1):23-36 [Non-Patent Document 3] Ellinwood et al., Mol Ther. 2011 Feb;19(2):251-9 [Non-Patent Document 4] Foust, et al., 2009, Nat.Biotechnol. 27:59-65 [Non-Patent Document 5] Duque et al., 2009, Mol.Ther. 17:1187-1196 [Non-Patent Document 6] Fu et al., 2011, Mol.Ther, 19:1025-1033 [Non-Patent Document 7] Gray et al., 2011, Mol.Ther. 19:1058-1069 [Non-Patent Document 8] Passini et al., 2014, Human Gene Therapy 25:619-630 [Non-Patent Document 9] Samaranch et al., 2012, Hum Gene Ther 23:382-389 Summary of the Invention

[0006] overview Compositions and methods are described for treating human subjects by administering a therapeutically effective amount of a single-stranded adeno-associated virus (ssAAV) vector or a self-complementary adeno-associated virus (scAAV) vector (e.g., a ssAAV9 vector or a scAAV9 vector) encoding a therapeutic gene product (e.g., a therapeutic protein or other therapeutic agent) by injecting the ssAAV vector or scAAV vector into the cerebrospinal fluid (CSF) via an intracerebroventricular (ICV) or intrathecal (cisternomagna or lumbar) route of administration. When an ICV route is utilized, bilateral ICV injections may be preferred in clinical situations.

[0007] The present invention is based, in part, on the inventors' discovery that ssAAV and scAAV vectors of the same serotype, such as AAV9, exhibit different cell tropism in the CNS when delivered to the CSF, which is surprising because, to the inventors' knowledge, ssAAV and scAAV vectors of the same serotype that exhibit different cell tropism have not been previously reported.

[0008] In some embodiments, scAAV9 is administered into the CSF to achieve superior transduction in the hippocampus, cerebellum, and cerebral cortex, where both neurons and glial cells (such as astrocytes) are transduced, with substantial transduction of cells in the cerebellum, particularly Purkinje neurons. This approach is particularly desirable when the transgene product is not secreted and thus does not result in cross correction of non-transformed cells. However, in diseases where astrocytosis and / or encephalitis are a concern, in specific, non-limiting examples, immunosuppressants are also administered.

[0009] For example, one embodiment includes delivery of a non-secreted therapeutic protein to the CNS of a human subject in need thereof, comprising administering to the human subject an effective amount of scAAV9 encoding the non-secreted therapeutic protein, wherein the scAAV9 is administered by injection into the CSF via intracerebroventricular, intrathecal cisternal, or intrathecal lumbar routes. A specific embodiment results in delivery of the therapeutic protein to neurons and glial cells of the brain. The method can further include administering a therapeutically effective amount of an immunosuppressant as a co-treatment. In certain embodiments, the therapeutic protein is not SMN1.

[0010] Another exemplary embodiment involves delivery of a therapeutic protein to neurons, Purkinje neurons, and / or astrocytes of a human subject in need thereof comprising administering to the human subject an effective amount of scAAV9 encoding the therapeutic protein, where the scAAV9 is administered by injection into the CSF via an intracerebroventricular, intrathecal cisternal, or intrathecal lumbar route. The method can further include administering a therapeutically effective amount of an immunosuppressant as a co-treatment.

[0011] In other embodiments, ssAAV9 is administered into the CSF to minimize transduction of astrocytes. In a specific, non-limiting example, ssAAV9 can be used to express secreted transgene products, in which case cross-correction can result in effective treatment of non-transformed CNS cells. In a specific, non-limiting example, an immunosuppressant is also administered.

[0012] Formulations of ssAAV and scAAV suitable for ICV delivery to human subjects, as well as therapeutically effective doses of ssAAV9 and scAAV9, are disclosed.

[0013] [The present invention 1001] administering to a human subject an effective amount of a self-complementary adeno-associated virus serotype 9 (scAAV9) encoding a non-secreted therapeutic protein. 1. A method for delivering a non-secreted therapeutic protein to the central nervous system (CNS) of a human subject in need thereof, comprising: [The present invention 1002] administering to the human subject an effective amount of a self-complementary adeno-associated virus serotype 9 (scAAV9) encoding a therapeutic protein. 1. A method for delivering a therapeutic protein to neurons, Purkinje neurons, and / or astrocytes of a human subject in need thereof, comprising: administering scAAV9 by injection into the cerebrospinal fluid (CSF) via an intraventricular, intrathecal cisternal, or intrathecal lumbar route. [The present invention 1003] The method of claim 1002, wherein the therapeutic protein is a non-secreted therapeutic protein. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the human subject is an infant. [The present invention 1005] Approximately 7.4×10 12 ~Approx. 1.86×10 14 The method of claim 1004, comprising administering a full dose of scAAV9 to a subject. [The present invention 1006] Any of the methods according to claims 1001 to 1003, wherein the subject is an adult. [The present invention 1007] Approximately 2.59×10 13 ~Approx. 6.51×10 14 The method of claim 1006, comprising administering a full dose of scAAV9 to the subject. [The present invention 1008] The method according to any one of claims 1001 to 1007, wherein the genetic disorder is a lysosomal storage disease. [The present invention 1009] The method of any of claims 1001 to 1008, comprising the step of administering a therapeutically effective amount of an immunosuppressant to a subject. [The present invention 1010] The method of any one of claims 10 to 15, wherein the immunosuppressant reduces the activity of cytotoxic T cells. [The present invention 1011] The method of any one of claims 1009 to 1010, wherein the immunosuppressant reduces encephalitis. [The present invention 1012] The method according to any one of claims 1009 to 1011, wherein the immunosuppressant is a nonsteroidal anti-inflammatory agent. [The present invention 1013] The method of any of claims 1001 to 1012, which reduces the risk of encephalitis in a subject. [The present invention 1014] The method of any of claims 1001 to 1013, wherein the scAAV9 transduces cells in the brain. [The present invention 1015] The method of any of claims 1001 to 1013, wherein the scAAV9 transduces cells in the hippocampus, cerebellum, and cerebral cortex. The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0014] [Figure 1] Figure 1a-1f. Extensive transduction of the CNS occurs after ICV scAAV9 delivery. Three weeks after receiving an ICV injection of scAAV9-GFP or saline, Sprague-Dawley rats were sacrificed. (a, b, d, e) Brain and spinal cord sections were stained for GFP (black dye). Sections from high-dose rats at approximately (a) 2.1 mm and (b) 11.3 mm caudal to the bregma are shown. Comparable regions from saline-treated rats are shown (d, e), revealing limited background staining. Notably, there was substantial transduction of (a) the cerebral cortex, hippocampus, and (b) the cerebellum. Transduction was also higher in the right side of the brain proximal to the injection tract. Deeper brain structures showed less transduction. (c) Transduction of the spinal cord was assessed by immunofluorescence (green dye). The anterior horn in the box in (c) is shown enlarged in (f). In contrast to the brain, there was little staining of cell bodies in the gray matter. Instead, the majority of the GFP staining appeared to be axons. Abbreviations: BS, brainstem; Cb, cerebellum; Cpu, caudate nucleus; Cx, cerebral cortex; Hp, hippocampus; T, thalamus. Scale bar in (f) represents 100 um. [Diagram 2]Figure 2a-2g. scAAV9 transduces several cell types when delivered by ICV injection in rats. (a) Brain sections from rats treated with scAAV9-GFP stained for GFP (black) show significant transduction of the choroid plexus (arrowhead) and ependyma (arrow), especially in the injected ventricle. (b-g) Brain sections were co-stained for GFP (green) and cell markers (red): (b) NeuN, (c) Fox2, (d) calbindin, (e) GFAP, (f) GAD67, and (g) S100β. Co-localization between GFP and cell markers is indicated by arrows. (b) Representative images from high-dose animals show co-localization of NeuN and GFP in hippocampal neurons. Co-localization with NeuN was also observed in the cerebral cortex. (c) Cerebellum of a low-dose animal is shown. Purkinje cells, identified by their elaborate dendritic arbors and the presence of Fox2 in the nuclei, were the predominant cells transduced. (d) Staining for calbindin, which stains a variety of neurons including Purkinje cells, also showed extensive colocalization with GFP (orange-yellow), supporting the conclusion that the majority of GFP expression in the cerebellum is due to transduction of Purkinje cells. GFP+ cells with a morphology similar to astrocytes were regularly observed in the cerebral cortex. (d) Staining for GFAP, a marker for astrocytes, and (e) GAD67, a marker for interneurons, failed to show colocalization with GFP for these cells. (f) However, S100β, a marker for a subset of astrocytes, stained most, if not all, of the cells with this morphology. [Figure 3A]Figure 3a-3c. Distribution of scAAV9 transduction. Transduction was quantified in three brain regions [(a) hippocampus, (b) cerebral cortex, and (c) cerebellum] in five treatment groups (PBS control, low dose, low dose / high volume, medium dose, and high dose). Each group consisted of three animals, except for the medium dose, which had two. Individual animals are plotted on the graph. Horizontal bars represent the mean for each group, and error bars show SEM. There was a general trend of greater transduction with increasing dose. Increasing injection volume did not appear to play a major role in the distribution of transduction. For the cerebral cortex and cerebellum, the medium and high dose groups were significantly different from the low dose and low dose / high volume groups (p<0.05). [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] Figure 4a-4f. scAAV9 transduction in pigs. Three weeks after ICV injection of scAAV9-GFP into one lateral ventricle, brains were harvested, sectioned, and stained for GFP (brown dye). Consistent with observations in rats, substantial transduction was observed in (a) the hippocampus, cerebral cortex, and (b) the cerebellum. Untreated pig brains show no staining for GFP (c). Photomicrographs taken at greater magnification are shown for (d) the cerebral cortex, (e) the hippocampus, and (f) the cerebellum. In (d), arrows indicate examples of cells with neuronal morphology. Arrowheads indicate examples of cells showing astrocytic morphology. The patterns of neuronal and glial transduction based on morphology are similar to those seen in rats. Scale bars are 100 um in (d) and (f) and 500 um in (e). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description For the potential treatment of disorders affecting the CNS by gene therapy, various administration routes for the delivery of viral vectors to the cerebrospinal fluid (CSF) (intrathecal, intracisternal, or intraventricular) have been investigated in animal models, with the goal of achieving transduction throughout the brain and spinal cord (CNS). Certain vectors, specifically adeno-associated virus serotype 9 (AAV9), have the ability to transduce many regions of the CNS after CSF delivery (Dayton et al., Expert Opin Biol Ther. 2012 Jun;12(6):757-66). In this regard, intracerebroventricular (ICV) delivery of AAV9 has been studied in neonatal or young mouse models to deliver genes to the CNS via the cerebrospinal fluid (CSF) (McClean et al., 2014, Neurosci.Lett. 576:73-8;Levites et al., J.Neurosci. 26:11923-8;Gholizadeh et al., 2013, 24:205-13;Dirren et al., 2014, Hum.Gene Ther. 25:109-20). However, the brain is not fully developed in these animal models, and marked differences in transduction patterns were observed when treatment ages were varied (Chakrabarty et al., 2013, PLoS One 8:e67680). It remains unclear how AAV9 functions in the adult brain, and most importantly, in human subjects. With the exception of one dog study (Haurigot et al., 2013, J. Clin. Invest. Jul 1), little is known about the biodistribution of AAV9 following ICV injection in animals larger than mice.

[0016] Indeed, Chakrabarty et al. (2013) demonstrated striking differences in transduction patterns when treatment ages were varied (9). Furthermore, the small size and resilience of newborn mice allows for the delivery of large doses that may not be practical in clinical situations. For example, one mouse study demonstrated approximately 3 × 10 14A dose of vector genome (vg) was administered (10) that was more than 100-fold higher than that used in a recent clinical trial for hemophilia, administered intravenously (11).

[0017] Prior to this disclosure, the biodistribution of AAV9 following ICV injection into animals larger than mice was largely unknown (12). Furthermore, the distribution of ICV self-complementary AAV9 (scAAV9), which is expected to provide superior transduction over traditional single-stranded AAV9 (ssAAV9), has not been investigated outside of neonatal mice. The dose-response relationship of ICV-delivered scAAV9 in adult rats was investigated using a range of doses that may be used clinically in human clinical trials. This study served as the basis for comparative studies in a large animal model, the domestic pig. The studies disclosed herein provide dosing decisions for humans, such as the use of ICV administration of ssAAV9 or scAAV9. The studies disclosed herein also demonstrate that ssAAV and scAAV vectors of the same serotype (ICV-delivered) exhibit different cell tropism in the CNS. This result was unexpected and forms the basis of the improved methods and compositions described herein for delivering therapeutic proteins to the CNS.

[0018] Specifically, scAAV9 achieves excellent transduction of both neuronal cells and glial cells, including astrocytes, in the hippocampus, cerebellum, and cerebral cortex.scAAV9 can be used to treat conditions where it is desirable to transduce all cells (including glial cells, such as astrocytes) to treat diseases (e.g., where the transgene product is not secreted and cross-collection does not occur or is unreliable).However, in certain diseases / conditions where astrocytosis / encephalitis is a concern, in some embodiments, immunosuppressants can also be administered.Thus, in some instances, immunosuppressive therapy can be used simultaneously to minimize the risk of side effects.

[0019] In additional embodiments, ssAAV9 can be used to treat diseases where central nervous system (CNS) inflammation / encephalitis is a concern, to transduce astrocytes and minimize induction of astrocytosis / encephalitis. In further embodiments, immunosuppressants can also be administered. In yet other embodiments, ssAAV9 treatment can be used for secreted transgene products where cross-correction can result in effective treatment of non-transformed CNS cells.

[0020] Dosages of ssAAV9 and scAAV9 and formulations that can be used for injection into the CSF in human subjects are as follows: For infants, about 4.4 x 10 13 ~Approx. 1.33×10 14 Full dose of GC (fixed dose) For adults, approximately 1.56 x 10 14 ~Approx. 4.67×10 14 Full dose of GC (fixed dose) For infants, approx. 7.4 x 10 12 ~Approx. 1.86×10 14 Full dose of GC (fixed dose) For adults, approximately 2.59 x 10 13 ~Approx. 6.51×10 14 Full dose of GC (fixed dose)

[0021] Thus, the dosage may be as follows: For infants, 4.4 x 10 13 ~1.33×10 14 Full dose of GC (fixed dose) For adults, 1.56 x 10 14 ~4.67×10 14 Full dose of GC (fixed dose) For infants, 7.4 x 10 12 ~1.86×10 14 Full dose of GC (fixed dose) For adults, 2.59 x 10 13 ~6.51×10 14 Full dose of GC (fixed dose)

[0022] Dosages for other uses are as follows: 3 to 9 months: Approx. 6.0 x 10 13 ~Approx. 1.9×10 14 GC; or approximately 1.1 × 10 14 ~Approx. 2.79×10 14 total dose of GC (fixed dose); 9 to 36 months: approx. 1.0 x 10 14 ~Approx. 3.3×10 14 GC; or approximately 1.85 × 10 13 ~Approx. 4.65×10 14 total dose of GC (fixed dose); Ages 3-12: Approx. 1.2 x 10 14 ~Approx. 3.96×10 14 GC; or approximately 2.2 × 10 13 ~Approx. 5.58×10 14 total dose of GC (fixed dose); Ages 12 and over: approx. 1.4 x 10 14 ~Approx. 4.62×10 14 GC; or approximately 2.59 × 10 13 GC~approx. 6.51×10 14 Full dose of GC (fixed dose).

[0023] Thus, the dosage for other uses is as follows: 3-9 months: 6.0×10 13 ~1.9×10 14 GC; or 1.1 × 10 14 ~2.79×10 14 total dose of GC (fixed dose); 9-36 months: 1.0×10 14 ~3.3×10 14 GC; or 1.85 × 10 13 ~4.65×10 14 total dose of GC (fixed dose); Ages 3-12: 1.2×10 14 ~3.96×10 14 GC; or 2.2 × 10 13 ~5.58×10 14 total dose of GC (fixed dose); Age 12 and over: 1.4×10 14 ~4.62×10 14GC; or 2.59 × 10 13 GC~6.51×10 14 Full dose of GC (fixed dose).

[0024] In another embodiment, the dose for a neonate is as follows: ·Approx. 4.4×10 13 ~Approx. 1.32×10 14 (fixed dose); or ·Approx. 7.4×10 12 ~Approx. 1.86×10 14 Total (fixed dose) GC.

[0025] Thus, the dose for a neonate might be as follows: 4.4×10 13 ~1.32×10 14 (fixed dose); or 7.4×10 12 ~1.86×10 14 Total (fixed dose) GC.

[0026] In a further embodiment, the dose is increased by two-fold, so the dose may be as follows: For infants, approx. 8.8 x 10 13 ~Approx. 2.66×10 14 Full dose of GC (fixed dose) For adults, approximately 3.21 x 10 14 ~Approx. 9.34×10 14 Full dose of GC (fixed dose) For infants, approx. 14.8 x 10 12 ~Approx. 3.72×10 14 Full dose of GC (fixed dose) For adults, approximately 5.18 x 10 13 ~Approx. 13.02×10 14 Full dose of GC (fixed dose)

[0027] Thus, the dosage may be as follows: For infants, 8.8 x 10 13 ~2.66×10 14 Full dose of GC (fixed dose) For adults, 3.21 x 1014 ~9.34×10 14 Full dose of GC (fixed dose) For infants, 14.8 x 10 12 ~3.72×10 14 Full dose of GC (fixed dose) For adults, 5.18 x 10 13 ~13.02×10 14 Full dose of GC (fixed dose)

[0028] Dosages for other uses are as follows: 3 to 9 months: Approx. 1.20 x 10 14 ~Approx. 3.8×10 14 GC; or approximately 2.2 × 10 14 ~Approx. 5.58×10 14 total dose of GC (fixed dose); 9-36 months: Approx. 2.0 x 10 14 ~Approx. 6.6×10 14 GC; or approximately 3.7 × 10 13 ~Approx. 9.3×10 14 total dose of GC (fixed dose); Ages 3-12: Approx. 2.4 x 10 14 ~Approx. 7.92×10 14 GC; or approximately 4.4 × 10 13 ~Approx. 1.116×10 15 total dose of GC (fixed dose); Ages 12 and over: Approx. 2.8 x 10 14 ~Approx. 9.24×10 14 GC; or approximately 5.18 × 10 13 GC~approx. 1.302×10 15 Full dose of GC (fixed dose).

[0029] Thus, the dosage for other uses is as follows: 3-9 months: 1.20 x 10 14 ~3.8×10 14 GC; or 2.2 × 10 14 ~5.58×10 14 total dose of GC (fixed dose); 9-36 months: 2.0×10 14 ~6.6×10 14 GC; or 3.7 × 1013 ~Approx. 9.3×10 14 total dose of GC (fixed dose); Ages 3-12: 2.4×10 14 ~7.92×10 14 GC; or 4.4 × 10 13 ~1.116×10 15 total dose of GC (fixed dose); Age 12 and over: 2.8×10 14 ~9.24×10 14 GC; or 5.18 × 10 13 GC~1.302×10 15 Full dose of GC (fixed dose).

[0030] In another embodiment, the dose for a neonate is: ·Approx. 8.8×10 13 ~Approx. 2.64×10 14 (fixed dose); or ·Approx. 1.48×10 13 ~Approx. 3.72×10 14 GC total (fixed dose) It is.

[0031] Thus, the dose for a neonate might be as follows: 8.8×10 13 ~2.64×10 14 (fixed dose); or 1.48×10 13 ~3.72×10 14 Total (fixed dose) GC.

[0032] In other embodiments, the dose is reduced and half of the original dose is utilized. Thus, the doses may be as follows: For infants, about 2.2 x 10 13 ~Approx. 6.65×10 13 Full dose of GC (fixed dose) For adults, approximately 7.8 x 10 13 ~Approx. 2.335×10 14 Full dose of GC (fixed dose) For infants, about 3.7 x 10 12 ~Approx. 9.3×10 13Full dose of GC (fixed dose) For adults, approximately 1.295 x 10 13 ~Approx. 3.225×10 14 Full dose of GC (fixed dose)

[0033] Thus, the dosage may be as follows: For infants, 2.2 x 10 13 ~6.65×10 13 Full dose of GC (fixed dose) For adults, 7.8 x 10 13 ~2.335×10 14 Full dose of GC (fixed dose) For infants, 3.7 x 10 12 ~9.3×10 13 Full dose of GC (fixed dose) For adults, 1.295 x 10 13 ~3.225×10 14 Full dose of GC (fixed dose)

[0034] Dosages for other uses are as follows: 3 to 9 months: Approx. 3.0 x 10 13 ~Approx. 9.5×10 13 GC; or approximately 5.5 × 10 13 ~Approx. 1.395×10 14 total dose of GC (fixed dose); 9-36 months: approx. 5 x 10 13 ~Approx. 1.65×10 14 GC; or approximately 9.25 × 10 12 ~Approx. 2.325×10 14 total dose of GC (fixed dose); Ages 3-12: Approx. 6 x 10 13 ~Approx. 1.98×10 14 GC; or approximately 1.1 × 10 13 ~Approx. 2.79×10 14 total dose of GC (fixed dose); Ages 12 and over: approx. 7 x 10 13 ~Approx. 2.31×10 14 GC; or approximately 1.295 × 10 13 GC~approx.3.225×10 14 Full dose of GC (fixed dose).

[0035] Thus, the dosage for other uses is as follows: 3 to 9 months: 3.0 x 10 13 ~9.5×10 13 GC; or 5.5 × 10 13 ~1.395×10 14 total dose of GC (fixed dose); 9-36 months: 5×10 13 ~1.65×10 14 GC; or 9.25 × 10 12 ~2.325×10 14 total dose of GC (fixed dose); Ages 3-12: 6×10 13 ~1.98×10 14 GC; or 1.1 × 10 13 ~2.79×10 14 total dose of GC (fixed dose); Age 12 and over: 7×10 13 ~2.31×10 14 GC; or 1.295 × 10 13 GC~3.225×10 14 Full dose of GC (fixed dose).

[0036] In another embodiment, the dose for a neonate is: ·Approx. 2.2×10 13 ~Approx. 6.6×10 13 (fixed dose); or ·Approx. 3.7×10 12 ~Approx. 9.3×10 13 GC total (fixed dose) It is.

[0037] Thus, the dose for a neonate might be as follows: 2.2×10 13 ~6.6×10 13 (fixed dose); or 3.7×10 12 ~9.3×10 13 Total (fixed dose) GC.

[0038] As indicated above with respect to dosage, "about" is within 5%. Note that genome content "GC" is equivalent to viral genome "vg". Those skilled in the art can easily determine GC. For example, GC can be determined by quantitative polymerase chain reaction (qPCR) using methods known in the art.

[0039] term Adeno-associated virus (AAV): AAV is a small virus that infects humans and several other primate species. AAV is not currently known to cause disease, and the virus induces a very mild immune response. AAV can infect both dividing and non-dividing cells and can integrate its genome into that of the host cell. The AAV genome is constructed from either positive- or negative-stranded single-stranded deoxyribonucleic acid (ssDNA) that is approximately 4.7 kilobases long. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and contains two open reading frames (ORFs): rep and cap. Rep is composed of four overlapping genes that code for the Rep proteins required for the AAV life cycle, and Cap contains overlapping nucleotide sequences of the capsid proteins: VP1, VP2, and VP3, which interact together to form a capsid with icosahedral symmetry. For gene therapy, the ITRs appear to be the only sequences required in cis next to the therapeutic gene: the structural (cap) and packaging (rep) genes can be delivered in trans. Currently, 11 serotypes of AAV (AAV1-11) are recognized.

[0040] Administration: Providing or giving agent to a subject by effective route.Exemplary administration routes include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.In some embodiments, administration is by injection into CSF ​​via intracerebroventricular (ICV) or intrathecal (cistema magna or lumbar) route.

[0041] Agent: Any polypeptide, compound, small molecule, organic compound, salt, polynucleotide, or other molecule of interest. Agents can include therapeutic agents, diagnostic agents, or drugs. A therapeutic agent is a substance that exhibits some therapeutic benefit by restoring or maintaining health, such as reducing symptoms associated with a disease or physiological disorder, or slowing (including preventing) the progression or onset of a disease.

[0042] Animal: A living multi-cellular vertebrate organism; this category includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. An "infant" is a human subject under the age of 1. An adult subject is one over the age of 18. A subject may be 3-9 months old, 9-36 months old, under 3 years old, 3-12 years old, or over 12 years old.

[0043] Antigen: A compound, composition, or substance capable of stimulating an immune response in an animal, such as the production of an antibody response or a T-cell response, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes.

[0044] Conservative substitution: Modifications of a polypeptide that involve the replacement of one or more amino acids with amino acids having similar biochemical properties, which do not result in a change or loss of the biological or biochemical function of the polypeptide, are referred to as "conservative" substitutions. These conservative substitutions are likely to have minimal effect on the activity of the resulting protein. Table 1 shows amino acids that can be substituted for the first amino acid in a protein and are considered conservative substitutions.

[0045] (table) TIFF2025041933000001.tif96128

[0046] One or more conservative changes, or up to ten conservative changes (e.g., two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, or five amino acid substitutions, etc.) can be made in a polypeptide without altering the biochemical function of the protein.

[0047] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence operably linked to it. An expression control sequence is operably linked to a nucleic acid sequence if it controls and regulates the transcription and, if appropriate, also controls and regulates the translation of the nucleic acid sequence. Thus, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, a start codon (i.e., ATG) in front of a gene encoding a protein, splicing signals of introns, maintaining the correct reading frame of the gene to allow proper translation of mRNA, and a stop codon. The term "control sequence" shall include at least components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. An expression control sequence may include a promoter. An expression control sequence may be operably linked to a nucleic acid molecule encoding a therapeutic protein and included in an AAV vector.

[0048] Heterologous: A heterologous sequence is a sequence that is not normally (in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the sequence is derived from a genetic source, such as a virus or organism, different from the second sequence.

[0049] Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be a prokaryotic or eukaryotic cell. The term also includes the progeny of the host cell. It is understood that all progeny may not be identical to the parent cell since mutations can occur during replication. However, such progeny are included when the term "host cell" is used.

[0050] Immune response: A response of a cell of the immune system, such as a B cell or a T cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response").

[0051] Immunosuppressant: A molecule, such as a chemical compound, small molecule, steroid, nucleic acid molecule, or other biological agent, that can reduce an immune response, such as an inflammatory response. Specific non-limiting examples of immunosuppressants are nonsteroidal anti-inflammatory agents, cyclosporine A, FK506, and anti-CD4. Immunosuppressant treatments may include prednisolone, mycophenolate mofetil (MMF), and sirolimus or tacrolimus. In additional examples, the agent is a biological response modifier such as KINERET® (anakinra), ENBREL® (etanercept), or REMICADE® (infliximab), a disease-modifying antirheumatic drug (DMARD) such as ARAVA® (leflunomide), a nonsteroidal anti-inflammatory drug (NSAID) such as a cyclooxygenase-2 (COX-2) inhibitor such as CELEBREX® (celecoxib) and VIOXX® (rofecoxib), or other products such as HYALGAN® (hyaluronic acid) and SYNVISC® (Hylan G-F20). In some embodiments, the immunosuppressant crosses the blood-brain barrier.

[0052] Isolated: An "isolated" biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, made separately from, or purified from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA and proteins, within the cells of the organism in which it naturally occurs. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. An isolated cell type is substantially separated from other cell types, such as different cell types present in an organ. A purified cell or component may be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0053] Label: A detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific non-limiting examples of labels include fluorescent tags, enzyme conjugations, and radioisotopes.

[0054] Lysosomal storage disease / disorder: Lysosomal storage disease / disorder is a type of disease that involves partial or complete deficiency of lysosomal hydrolases. This deficiency leads to incomplete lysosomal digestion of substrates specific for the hydrolases. Over time, the accumulation of undigested substrates can lead to a variety of abnormalities, including progressive and severe neurodegeneration and muscular degeneration (see Settembre et al., Human Mol.Genet., 17:119-129,2008; Fukuda et al., Curr.Neurol.Neurosci.Rep., 7:71-77,2007). The term lysosomal storage disorder is synonymous with lysosomal storage disease.

[0055] Lysosomal storage disorders include GM2 gangliosidosis, alpha-mannosidosis, aspartylglucosaminuria, cholesteryl ester storage disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease, GM1 gangliosidosis, I cell disease / mucolipidosis II, infantile free sialic acid storage disease / ISSD, juvenile hexosaminidase A deficiency, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidoses (pseudo-Hurler-polydystrophy / mucolipidosis IIIA, MPS I-Hurler syndrome, MPS I-Scheie syndrome, MPS I-Hurler-Scheie syndrome, MPS II-Hunter syndrome, Sanfilippo syndrome, Morquio A / MPS IVA, Morquio B / MPS IVB, MPS IX hyaluronidase deficiency, MPS Malotheus-Lamyi, MPS VII Sly syndrome, Mucolipidosis I / sialidosis, Mucolipidosis IIIC, Mucolipidosis IV), Multiple sulfatase deficiency, Niemann-Pick disease, Neuronal ceroid lipofuscinosis (CLN6 disease, Batten-Spielmeier-Voigt / Juvenile NCL / CLN3 disease, Finnish variant late infantile CLN5, Jansky-Bielschowski disease / Late infantile CLN2 / TPP1 disease, Kuchs / Adult-onset NCL / CLN4 disease, Northern epilepsy (Northern These include, but are not limited to, late infantile CLN8 / variant epilepsy, Santavuori-Haltia / infantile CLN1 / PPT disease, beta-mannosidosis, Pompe disease / glycogen storage disease type II, pyknodysostosis, Sandhoff disease, Schindler disease, Salla disease / sialic acid storage disease, Tay-Sachs / GM2 gangliosidosis, and Wolman disease.

[0056] Non-lysosomal storage disorders: Non-lysosomal storage disorders that may be treated using the methods disclosed herein include spinal muscular atrophy (SMA) types 1, II, III, IV (SMN1 gene, UBA1 gene, DYNC1H1 gene, VAPB gene), X-linked myotubular myopathy (MTM1 gene), catecholamine-induced polymorphic ventricular tachycardia (CASQ2 gene), color vision disorders (CNGB3 gene, CNGA3 gene, GNAT2 gene, PDE6C gene), total choroidal atrophy (CHM gene), Friedreich's ataxia CNS (FXN gene), child), generalized Friedreich's ataxia (FXN gene), adrenoleukodystrophy (ABCD1 gene), Alzheimer's disease (APP gene, PPARγ gene), amyotrophic lateral sclerosis (SOD1 gene), Angelman syndrome (UBE3A gene), ataxia telangiectasia (ATM gene), Charcot-Marie-Tooth syndrome (PMP22 gene), Cockayne syndrome (ERCC6 gene, ERCC8 gene), hearing loss (GJB2 gene), Duchenne muscular dystrophy (DMD gene), epilepsy (SCN1A gene) ), Fragile X syndrome (FMR1 gene), Huntington's disease (HTT gene), Lesch-Nyhan syndrome (HGPRT gene), Maple syrup urine disease (BCKDHA gene, BCKDHB gene, DBT gene), Menkes syndrome (ATP7A gene), Myotonic dystrophy (DMPK), Narcolepsy (HLA gene), Neurofibromatosis (NF1 gene), Parkinson's disease (LRRK2 gene, PARK2 gene, PARK7 gene, PINK1 gene, SNCA gene), Phenylketonuria (PAH gene), Prader-Williams syndrome (PWB gene), These include, but are not limited to, Johns Hopkins syndrome, Refsum disease (PEX7 gene), Rett syndrome (MECP2 gene), spinocerebellar ataxia (SCA1 gene), Tangier disease (ABCA1 gene), tuberous sclerosis complex (TSC1 gene, TSC2 gene), von Hippel-Lindau syndrome (VHL gene), Williams syndrome (CLIP2 gene, ELN gene, GTF2I gene, GTF2IRD1 gene, LIMK1 gene), Wilson's disease (ATP7B gene), or Zellweger syndrome (PEX1 gene).

[0057] Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

[0058] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and the bonds between them include non-naturally occurring synthetic analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no more than about 50 nucleotides. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences (i.e., A, U, G, C) in which "T" is replaced by "U".

[0059] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5'-end; the left-hand end of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having a sequence identical to the mRNA is referred to as the "coding strand"; the sequence on the DNA strand having a sequence identical to the mRNA transcribed from that DNA and located 5' to the 5'-end of the RNA transcript is referred to as the "upstream sequence"; the sequence on the DNA strand having a sequence identical to the RNA and located 3' to the 3'-end of the coding RNA transcript is referred to as the "downstream sequence".

[0060] "cDNA" refers to a DNA that is complementary or identical to an mRNA in either single-stranded or double-stranded form.

[0061] "Encoding" refers to the inherent property of a particular nucleotide sequence of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or with a defined amino acid sequence, and the biological properties attributed thereto. Thus, if transcription and translation of the mRNA produced by a gene produces a protein in a cell or other biological system, the gene codes for that protein. Both the coding sequence of a gene or cDNA, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription, can be said to code for the protein or other product of the gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and code for the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can include introns.

[0062] "Recombinant nucleic acid" refers to a nucleic acid having a nucleotide sequence that is not joined together in nature. It includes a nucleic acid vector containing an amplified or assembled nucleic acid that can be used to transform an appropriate host cell. A host cell containing a recombinant nucleic acid is called a "recombinant host cell." Genes are then expressed in the recombinant host cell to produce "recombinant polypeptides," etc. Recombinant nucleic acids can also serve non-coding functions (such as promoters, origins of replication, ribosome binding sites, etc.).

[0063] A polynucleotide having a first sequence is "antisense" to a polynucleotide having a second sequence when the first sequence specifically hybridizes to the polynucleotide having the second sequence.

[0064] Terms used to describe sequence relationships between two or more nucleotide or amino acid sequences include "reference sequence," "selected from," "comparison window," "identical," "percentage of sequence identity," "substantially identical," "complementary," and "substantially complementary."

[0065] For the sequence comparison of nucleic acid sequences, typically, one sequence acts as a reference sequence to which test sequences are compared.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters are used.Methods for alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1998, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)).

[0066] One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35: 351-360, 1987. The method used is similar to that described by Higgins & Sharp, CABIOS 5: 151-153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine sequence identity (%) relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, such as version 7.0 (Devereaux et al., Nuc. Acids Res. 12: 387-395, 1984).

[0067] Another example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms described in Altschul et al., J.Mol.Biol. 215:403-410,1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignment (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

[0068] Functionally linked: A first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is functionally linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0069] ORF (Open Reading Frame): A series of nucleotide triplets (codons) that code for amino acids without a termination codon. These sequences are generally translatable into a peptide.

[0070] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers useful in the present invention are conventional. Remington's Pharmaceutical Sciences by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and formulations suitable for pharmaceutical delivery of the AAV vectors disclosed herein.

[0071] Generally, the nature of the carrier will depend on the particular mode of administration to be employed. For example, parenteral formulations generally contain injectable fluids that contain pharma- ceutically and physiologically acceptable liquids as vehicles, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. Conventional non-toxic solid carriers for solid compositions (e.g., in powder, pill, tablet, or capsule forms) can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0072] Agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or cell. "Incubating" includes a period of time sufficient for the drug to interact with the cells. "Contacting" includes incubating a drug in solid or liquid form with the cells.

[0073] Polypeptide: A polymer in which the monomers are amino acid residues joined together through amide bonds. When the amino acids are alpha amino acids, either the L-optical isomer or the D-optical isomer may be used, with the L-isomer being preferred. The term "polypeptide" or "protein" as used herein is intended to encompass any amino acid sequence, including modified sequences such as glycoproteins. The term "polypeptide" specifically encompasses naturally occurring proteins, and is also intended to encompass those produced recombinantly or synthetically.

[0074] The term "polypeptide fragment" refers to a portion of a polypeptide that exhibits at least one useful epitope. The term "functional fragment of a polypeptide" refers to all fragments of a polypeptide that retain the activity of the polypeptide. Biologically functional fragments can vary in size from small polypeptide fragments, such as epitopes that can bind to antibody molecules, to larger polypeptides that can participate in the characteristic induction or programming of phenotypic changes in cells. An "epitope" is a region of a polypeptide that can bind to immunoglobulins that are generated in response to contact with an antigen. Thus, smaller peptides that contain the biological activity of insulin, or conservative variants of insulin, are included as being useful.

[0075] The term "soluble" refers to a form of a polypeptide that is not inserted into a cell membrane.

[0076] The term "substantially purified polypeptide," as used herein, refers to a polypeptide that is substantially free from other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50%, such as at least 80%, free from other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free from other proteins, lipids, carbohydrates, or other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free from other proteins, lipids, carbohydrates, or other materials with which it is naturally associated.

[0077] A conservative substitution is the exchange of one amino acid with another that is similar in size, hydrophobicity, etc. Variations in the cDNA sequence that result in amino acid changes, whether conservative or not, should be minimized to preserve the functional identity and immunological identity of the encoded protein. The immunological identity of a protein can be assessed by determining whether it is recognized by an antibody; a variant that is recognized by such an antibody is immunologically conserved. A cDNA sequence variant will preferably introduce no more than 20 amino acid substitutions, preferably less than 10, into the encoded polypeptide. A variant amino acid sequence can be, for example, 80%, 90%, or even 95% or 98% identical to the native amino acid sequence.

[0078] Polynucleotide: A nucleic acid sequence of any length (such as a linear sequence). Thus, polynucleotide includes oligonucleotides, including gene sequences found in chromosomes. An "oligonucleotide" is a plurality of joined nucleotides joined by native phosphodiester bonds. An oligonucleotide is a polynucleotide that is 6-300 nucleotides in length. An oligonucleotide analog refers to a moiety that has a similar function to an oligonucleotide, but has non-naturally occurring portions. For example, an oligonucleotide analog may contain non-naturally occurring portions, such as modified sugar moieties or inter-sugar linkages, such as phosphorothioate oligodeoxynucleotides. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.

[0079] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the onset of the disease altogether. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after onset. "Amelioration" refers to a reduction in the number or severity of signs or symptoms of a disease.

[0080] Promoter: A promoter is an array of nucleic acid control sequences that directs the transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the transcription start site, such as a TATA element in the case of a polymerase II type promoter. A promoter also includes a distal enhancer or repressor element, which may be located as far away as several thousand base pairs from the start site of transcription. It also includes sufficient promoter elements to make promoter-dependent gene expression cell type-specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987).

[0081] Purified: The term "purified" does not require absolute purity; rather, it is intended to be a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that has been completely or partially isolated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the primary preparation, such as proteins, cellular debris, and other components.

[0082] Recombinant: A recombinant nucleic acid is one that has a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separated sequence segments. This artificial combination is often achieved by the artificial manipulation of isolated segments of nucleic acid, such as chemical synthesis or, more commonly, genetic engineering techniques. Similarly, a recombinant protein is one that is encoded by a recombinant nucleic acid molecule. Furthermore, a recombinant virus is a virus that contains a sequence (such as a genomic sequence) that does not occur in nature or that is created by the artificial combination of at least two sequences of different origins. The term "recombinant" also includes nucleic acids, proteins, and viruses that are altered only by the addition, substitution, or deletion of a portion of a naturally occurring nucleic acid molecule, protein, or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule (such as a recombinant nucleic acid molecule encoding a therapeutic protein) is packaged.

[0083] Selectable marker: A gene, RNA, or protein that confers a selectable phenotype to a cell, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), prototrophy, or the expression of a specific protein that can be used as a basis for distinguishing cells that express the protein from cells that do not. Proteins whose expression can be easily detected ("detectable markers"), such as fluorescent or luminescent proteins or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance, constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to genes that are normally expressed selectively or exclusively in pluripotent cells allows for the identification and selection of specific cells of interest. A variety of selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue fluorescent protein, sapphire fluorescent protein, yellow fluorescent protein, red fluorescent protein, orange fluorescent protein, and cyan fluorescent protein, and variants of any of these. Luminescent proteins, such as luciferase (e.g., firefly luciferase or Renilla luciferase), are also useful. The term "selectable marker" as used herein can refer to a gene or the expression product of a gene, such as an encoded protein.

[0084] Sequence identity of amino acid sequences: Similarity between amino acid sequences is expressed by the similarity between sequences, also called sequence identity. Sequence identity is often measured by the percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0085] The method of aligning sequences for comparison is well known in the art.Various programs and alignment algorithms are described in Smith and Waterman, Adv.Appl.Math. 2:482, 1981;Needleman and Wunsch, J.Mol.Biol. 48:443, 1970;Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444, 1988;Higgins and Sharp, Gene 73:237, 1988;Higgins and Sharp, CABIOS 5:151, 1989;Corpet et al., Nucleic Acids Research 16:10881, 1988;and Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0086] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD), or on the Internet. Instructions on how to use this program to determine sequence identity are available on the Internet at the NCBI website.

[0087] Protein homologs and variants are typically characterized by having at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, as counted in full-length alignments with the amino acid sequence of the antibody, using NCBI Blast 2.0, gapped blastp, set to default parameters. For comparison of amino acid sequences of more than about 30 amino acids, the Blast 2 sequences function is utilized using the BLOSUM62 matrix set to default parameters (gap existence cost 11, gap cost per residue 1). When aligning short peptides (less than about 30 amino acids), the alignment should be performed using the Blast2 sequences function, using the PAM30 matrix set to default parameters (open gap penalty 9, extension gap penalty 1). Proteins with even greater similarity to the reference sequence, when assessed by this method, will show increasing percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than full-length sequences are compared for sequence identity, homologs and variants typically share at least 80% sequence identity in a short window of 10-20 amino acids, and may share at least 85% or at least 90% or 95% identity, depending on the similarity to the reference sequence. Methods for determining sequence identity in such short windows are available on the Internet at the NCBI website. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is also possible that strongly significant homologs are available that do not fall within the provided ranges.

[0088] Subject: A mammal, such as a human, non-human primate, pig, sheep, cow, rodent, etc., that is amenable to a particular treatment. In two non-limiting examples, the subject is a human subject or a murine subject.

[0089] Therapeutic agent: used in a comprehensive sense, including treatment agents, prophylactic agents, and supplemental agents. Therapeutic agents can be therapeutic proteins. Therapeutic agents, such as therapeutic proteins, can be delivered to a subject to prevent, treat, and / or ameliorate disease. The disease can be a disease of the central nervous system, such as a lysosomal storage disorder or a non-lysosomal storage disorder.

[0090] Therapeutically effective amount: An amount of a particular drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve a desired effect, such as increased insulin production, in a subject or cell being treated with the agent. The effective amount of the agent will depend on several factors, including, but not limited to, the subject or cell being treated and the mode of administration of the therapeutic composition.

[0091] Transduced and transformed: A virus or vector "transduces" a cell when it transfers nucleic acid to the cell. A cell has been "transformed" or "transfected" by a nucleic acid that has been transduced into the cell when the DNA is stably replicated by the cell, either by integration of the nucleic acid into the cell's genome or by episomal replication.

[0092] Many methods of transfection are known to those skilled in the art, such as chemical methods (e.g., calcium phosphate transfection), physical methods (e.g., electroporation, microinjection, biolistics), fusion (e.g., liposomes), receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope / capsid-DNA complexes), and biological infection with viruses, such as recombinant viruses (Wolff, JA ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)). In the case of infection with retroviruses, infectious retroviral particles are absorbed into the target cell, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting provirus into cellular DNA. Methods of gene introduction into pancreatic endocrine cells are known (see, for example, U.S. Patent No. 6,110,743, which is incorporated herein by reference). These methods can be used to transduce pancreatic endocrine cells produced by the methods described herein or artificial islets produced by the methods described herein.

[0093] Genetic modification of a target cell is evidence of successful transfection. A "genetically modified cell" refers to a cell whose genotype has been altered as a result of cellular uptake of an exogenous nucleotide sequence by transfection. Reference to a transfected or genetically modified cell includes both the particular cell into which the vector or polynucleotide has been introduced and the progeny of that cell.

[0094] Transgene: A foreign gene delivered by a vector.

[0095] Vector: A nucleic acid molecule that is introduced into a host cell, thereby resulting in a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more therapeutic genes and / or selectable marker genes, as well as other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally contains materials that help achieve entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating, etc. In some embodiments herein, the vector is an AAV vector.

[0096] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. "Comprising A or B" means including A, or B, or A and B. Furthermore, all base or amino acid sizes and all molecular weight or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0097] ssAAV9 vector Disclosed herein are methods and compositions that include one or more vectors, such as viral vectors, e.g., adenoviral vectors or AAV. Defective viruses that completely or almost completely lack viral genes can be used. The use of defective viral vectors allows for administration to specific cells without the concern that the vector will infect other cells. Useful adenoviral and AAV vectors include replication-competent, replication-defective, and gutless types. Without being bound by theory, adenoviral vectors are known to exhibit strong expression in vitro, excellent titers, and the ability to transduce dividing and non-dividing cells in vivo (Hitt et al., Adv in Virus Res 55:479-505, 2000). When used in vivo, these vectors result in strong but transient gene expression due to the immune response elicited against the vector backbone. In some non-limiting examples, useful vectors are attenuated adenovirus vectors, such as those described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 1992; La Salle et al., Science 259:988-990, 1993); or defective AAV vectors (Samulski et al., J. Virol., 61:3096-3101, 1987; Samulski et al., J. Virol., 63:3822-3828, 1989; Lebkowski et al., Mol. Cell Biol., 8:3988-3996, 1988).

[0098] Recombinant AAV vectors are characterized by their ability to direct the expression and production of a selected transgenic product in a target cell, and thus contain at least all of the AAV sequences essential for encapsidation and the physical structure for infection of a target cell.

[0099] AAV belongs to the Dependovirus genus of the Parvoviridae family. AAV is a small non-enveloped virus that packages a linear single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into AAV capsids at equal frequency. In some embodiments, the AAV DNA comprises a nucleic acid encoding a therapeutic protein. Recombinant vectors, such as recombinant adenovirus vectors and recombinant adeno-associated virus (rAAV) vectors, comprising the nucleic acid molecules disclosed herein are further provided. In some embodiments, the AAV is AAV9.

[0100] The AAV genome features two inverted terminal repeats (ITRs) flanking two open reading frames (ORFs). In the AAV2 genome, for example, the first 125 nucleotides of the ITR are palindromic and fold back on themselves to maximize base pairing, forming a T-shaped hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. The ITRs are cis-acting sequences important for AAV DNA replication; they are the origin of replication and serve as primers for second-strand synthesis by DNA polymerase. The double-stranded DNA formed in this synthesis is called replicative monomer and is used for a second round of self-priming replication to form replicative dimers. These double-stranded intermediates are processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Rep binding elements and terminal release sites (TRS) are located within the ITRs. These features are used by the viral regulatory protein Rep during AAV replication to process the double-stranded intermediates. In addition to their role in AAV replication, ITRs are also essential for packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration of the AAV genome (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008). In some embodiments, these elements are included in the AAV vector.

[0101] The left ORF of AAV contains the Rep gene, which encodes four proteins: Rep78, Rep68, Rep52, and Rep40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP3). The AAV capsid contains 60 viral capsid proteins arranged in an icosahedral symmetry. VP1, VP2, and VP3 are present in a molar ratio of 1:1:10 (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008). In some embodiments, these elements are included in an AAV vector, such as an AAV9 vector.

[0102] AAV infects humans and some other primate species, but is not known to cause disease and induces a very mild immune response.AAV-based gene therapy vectors infect both dividing and quiescent cells and persist in an extrachromosomal state without being integrated into the genome of host cells.AAV possesses several additional desirable features for gene therapy vectors, including the ability to bind and invade target cells, enter the nucleus, be expressed in the nucleus for a long period of time, and low toxicity.AAV can be used to transfect cells, and suitable vectors are known in the art (see, for example, U.S. Patent Application Publication No. 2014 / 0037585, which is incorporated herein by reference). Methods for generating rAAV suitable for gene therapy are well known in the art (see, e.g., U.S. Published Patent Applications Nos. 2012 / 0100606; 2012 / 0135515; 2011 / 0229971; and 2013 / 0072548; and Ghosh et al., Gene Ther 13(4):321-329, 2006) and can be utilized in the methods disclosed herein.

[0103] A vector useful in the methods disclosed herein may contain a nucleic acid sequence encoding a complete AAV capsid, which may be derived from a single AAV serotype (e.g., AAV9, as disclosed in WO2005 / 033321, which is incorporated herein by reference). As disclosed in U.S. Patent No. 8,692,332, a useful vector may be recombinant and thus contain a sequence encoding an artificial capsid containing one or more fragments of the AAV9 capsid fused to a heterologous AAV or non-AAV capsid protein (or fragments thereof). These artificial capsid proteins are selected from non-contiguous portions of the AAV9 capsid or capsids of other AAV serotypes. For example, a rAAV vector may have a capsid protein that includes an AAV9 capsid region selected from VP2 and / or VP3 or VP1, or one or more of fragments thereof selected from amino acids 1-184, amino acids 199-259; amino acids 274-446; amino acids 603-659; amino acids 670-706; amino acids 724-738 of the AAV9 capsid. In another example, it may be desirable to change the start codon of the VP3 protein to GTG. Alternatively, the rAAV may contain one or more of the AAV serotype 9 capsid protein hypervariable regions (see, e.g., WO 2005 / 033321, incorporated herein by reference).

[0104] In some embodiments, a recombinant adeno-associated virus (rAAV) having an AAV serotype 9 capsid is generated. To generate the vector, a host cell can be cultured that contains a nucleic acid sequence encoding an AAV serotype 9 capsid protein as defined herein, or a fragment thereof; a functional rep gene; a minigene consisting of at least an AAV inverted terminal repeat (ITR) and a transgene, such as a transgene encoding a therapeutic protein; and sufficient helper functions to allow packaging in an AAV8 capsid protein. The components required to be cultured in a host cell for packaging an AAV minigene into an AAV capsid may be provided in trans to the host cell. Alternatively, one or more of the required components (e.g., a minigene, a rep sequence, a cap sequence, and / or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the required components using methods known to those skilled in the art. In some embodiments, the stable host cell will contain the required components under the control of an inducible promoter. However, the required components may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are known in the art.

[0105] In yet another alternative, the selected stable host cell may contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain the rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells can be generated by one of skill in the art.

[0106] The minigene, rep sequence, cap sequence, and helper functions required to make rAAV can be delivered to the packaging host cell in the form of any genetic element that transfers the retained sequences. The selected genetic element can be delivered by any suitable method, including the methods described herein. The methods used to construct the vector are known to those skilled in the art of nucleic acid manipulation and include genetic modification, recombinant modification, and synthetic techniques. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation of the present invention. See, for example, K.Fisher et al., J.Virol., 70:520-532 (1993), U.S. Patent No. 5,478,745, and PCT Publication No. WO2005 / 033321, which are incorporated herein by reference. In some embodiments, selected AAV components can be readily isolated from AAV serotypes, including AAV9, using techniques available to those of skill in the art. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences can be obtained through synthesis or other suitable means, by reference to published sequences such as those available in the literature or databases such as GENBANK®.

[0107] scAAV vectors scAAV vectors are disclosed in McCarty et al., 2001, Gene Ther. 8:1248-1254; Carter, PCT Publication No. WO 2001 / 011034; and Samulski, PCT Publication No. WO 2001 / 092551, all of which are incorporated herein by reference.

[0108] As disclosed in PCT Publication No. "double-stranded" DNA parvovirus vectors (as described above) can be advantageously utilized for gene delivery. Double-stranded parvoviruses can provide improved transducing to particle ratios, more rapid transgene expression, higher levels of transgene expression, and / or more sustained transgene expression. Double-stranded parvovirus vectors can be used for gene delivery into host cells that are typically resistant to AAV transduction. Thus, double-stranded parvovirus vectors such as AAV9 have a different host range than ssAAV vectors.

[0109] These vectors are dimeric self-complementary (sc) polynucleotides (typically DNA) packaged into a viral capsid, such as a parvovirus capsid, e.g., an AAV capsid, such as AAV9. In some respects, the viral genome packaged into the capsid is essentially a "trapped" replicative intermediate that cannot dissociate to produce the parvovirus DNA strands of positive and negative polarity. Thus, double-stranded parvovirus vectors can avoid the need for host cell-mediated synthesis of complementary DNA, which is inherent in conventional recombinant AAV (ssAAV) vectors.

[0110] This result is achieved by allowing the virus to package essentially dimeric inverted repeats of single-stranded parvovirus (e.g., ssAAV, such as ssAAV9) vector genomes such that both strands joined at one end are contained in a single infectious capsid. Upon release from the capsid, the complementary sequences reanneal within the target cell to form transcriptionally active double-stranded DNA. Duplex parvovirus vectors are fundamentally different from conventional parvovirus (e.g., ssAAV, such as ssAAV9) vectors and parent parvoviruses (e.g., AAV, such as AAV9) in that the vDNA can form a double-stranded hairpin structure due to intrastrand base pairing, and both DNA strands are encapsidated. Thus, duplex parvovirus vectors are functionally similar to double-stranded DNA viral vectors rather than the parvoviruses (e.g., ssAAV, such as AAV9) from which they are derived.

[0111] The viral capsid can be derived from any parvovirus, either autonomous parvovirus or dependovirus.Preferably, the viral capsid is an AAV capsid (e.g., AAV9 capsid).The selection of the parvovirus capsid can be based on a number of considerations known in the art, such as the type of target cell, the desired expression level, the nature of the heterologous nucleotide sequence expressed, the challenges associated with virus production, etc.In a specific example, the capsid is an AAV9 capsid.

[0112] Parvovirus particles may be "hybrid" particles in which the viral TRs and viral capsids are derived from different parvoviruses. Preferably, the viral TRs and capsids are derived from different serotypes of AAV (e.g., as described in International Patent Publication WO00 / 28004, US Provisional Application No. 60 / 248,920; and Chao et al., (2000) Molecular Therapy 2:619, the disclosures of which are incorporated herein in their entirety). Similarly, parvoviruses may have "chimeric" capsids (e.g., containing sequences derived from different parvoviruses) or "targeted" capsids (e.g., directional tropism) as described in these publications. As used herein, "double-stranded parvovirus particles" encompass hybrid, chimeric, and targeted viral particles. Preferably, double-stranded parvovirus particles have AAV capsids, which may further be chimeric or targeted capsids as described above.

[0113] The double-stranded parvovirus vector can be produced by any suitable method. Preferably, the template for producing vDNA is one that preferentially produces duplexes (i.e., the majority of vDNA produced is double-stranded) rather than monomeric vDNA capable of forming double-stranded vDNA. In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more of the replication products from the template are double-stranded. In one specific embodiment, the template is a DNA molecule that includes one or more terminal repeat (TR) sequences. The template includes a modified TR that cannot be dissociated (i.e., cannot be nicked) by the parvovirus Rep protein. During replication, the inability of the Rep protein to dissociate the modified TR will result in a stable intermediate in which two "monomers" are covalently attached by the non-degradable TR. This "duplexed" molecule can be packaged into a parvovirus (AAV) capsid to generate novel duplexed parvovirus vectors, such as scAAV9 vectors.

[0114] Without wishing to be bound by any particular theory, it is likely that the virion genome is maintained in a single-stranded form while packaged within the viral capsid. Upon release from the capsid during viral infection, the dimeric molecules "snap back" or anneal to form a double-stranded molecule by intrastrand base pairing, with the non-releasable TR sequence at one end forming a covalently closed hairpin structure. This double-stranded vDNA dispenses with the host cell-mediated second strand synthesis, which is hypothesized to be the rate-limiting step for AAV transduction.

[0115] In some embodiments, the template further comprises a heterologous nucleotide sequence (described below) that is packaged for delivery to a target cell. According to this specific embodiment, the heterologous nucleotide sequence is located between the viral TRs at either end of the substrate. In a further preferred embodiment, the parvovirus (e.g., AAV) cap gene and the parvovirus (e.g., AAV) rep gene are deleted from the template (and the vDNA produced therefrom). This configuration maximizes the size of the heterologous nucleic acid sequence that the parvovirus capsid can carry.

[0116] In one specific embodiment, the template for generating a double-stranded parvovirus vector contains at least one TR at the 5' and 3' ends flanking the heterologous nucleotide sequence of interest (described below). The TR at one end of the substrate is non-releasable, i.e., cannot be dissociated (nicked) by the Rep protein. During replication, the inability of the Rep protein to dissociate one of the TRs will result in a stable intermediate with two "monomers" covalently attached by a non-functional (i.e., non-releasable) TR. The heterologous nucleotide sequence can be in either orientation relative to the non-releasable TR.

[0117] The term "adjacent" does not necessarily indicate that the sequences are contiguous. For example, in the example of the previous paragraph, there may be an intervening sequence between the heterologous nucleotide sequence and the TR. A sequence that is "adjacent" to two other elements indicates that one element is located 5' to the sequence and the other element is located 3' to the sequence; however, there may be an intervening sequence between them.

[0118] According to this embodiment, the template for producing double-stranded parvovirus vDNA is about half the size of the wild-type parvovirus genome (e.g., AAV) corresponding to the capsid in which the vDNA is packaged. In other words, the template is preferably about 40% to about 55% by weight, more preferably about 45% to about 52% by weight. Thus, the double-stranded vDNA produced from this template is preferably about the size of the wild-type parvovirus genome (e.g., AAV) corresponding to the capsid in which the vDNA is packaged, for example, about 80% to about 105% by weight. In the case of AAV, it is well known in the art that AAV capsids are inconvenient for packaging vDNA whose size substantially deviates from the wt AAV genome. In the case of AAV capsids, the template is preferably about 5.2 kb or less in size. In other embodiments, the template is preferably greater than about 3.6 kb, 3.8 kb, 4.0 kb, 4.2 kb, or 4.4 kb in length, and / or less than about 5.4 kb, 5.2 kb, 5.0 kb, or 4.8 kb in length. In other words, the heterologous nucleotide sequence is typically less than about 2.5 kb in length (more preferably less than about 2.4 kb, even more preferably less than about 2.2 kb, even more preferably less than about 2.1 kb in length) to facilitate packaging of the double-stranded template by the parvovirus (e.g., AAV) capsid. In another specific embodiment, the template itself is a double-stranded, i.e., dimeric, self-complementary molecule. According to this embodiment, the template comprises a releasable TR at either end. The template further comprises a centrally located non-releasable TR (as described above). In other words, each half of the template on either side of the non-releasable TR is approximately the same length. Each half of the template (i.e., between the releasable and non-releasable TRs) contains one or more heterologous nucleotide sequences of interest. The heterologous nucleotide sequences in each half of the molecule are flanked by releasable TRs and a central non-releasable TR.

[0119] The sequences of either half of the template are substantially complementary (i.e., at least about 90%, 95%, 98%, 99%, or more nucleotide sequence complementarity) such that the products of replication from the template can form a double-stranded molecule by base pairing between the complementary sequences. In other words, the template is essentially an inverted repeat in which the two halves are joined by a non-releasable TR.

[0120] In some non-limiting examples, the heterologous nucleotide sequence of each half of the template is essentially completely self-complementary (i.e., contains few or no mismatched bases). In additional non-limiting examples, the two halves of the nucleotide sequence are essentially completely self-complementary.

[0121] According to this embodiment, the template (and vDNA made therefrom) is preferably about the same size as the wt genome naturally enveloped by the parvovirus capsid (e.g., AAV), i.e., to facilitate efficient packaging into the parvovirus capsid. For example, in the case of an AAV capsid, the template is preferably about the size of the wild-type (wt) AAV genome. In a specific embodiment, the template is about 5.2 kb or less in size. In other embodiments, the template is preferably greater than about 3.6 kb, 3.8 kb, 4.0 kb, 4.2 kb, or 4.4 kb in length, and / or less than about 5.4 kb, 5.2 kb, 5.0 kb, or 4.8 kb in length. In other words, the template is preferably in the range of 80% to 105% of the wild-type parvovirus genome (e.g., AAV).

[0122] The TRs (releasable and non-releasable) are preferably AAV sequences, with serotypes 1, 2, 3, 4, 5, and 6 being preferred. The term "terminal repeat" includes synthetic sequences that function as AAV inverted terminal sequences, such as the "double D sequence" as described in U.S. Patent No. 5,478,745 to Samulski et al., the entire disclosure of which is incorporated herein by reference. The releasable AAV TRs may not have wild-type TR sequences (e.g., the wild-type sequence may be altered by insertion, deletion, truncation, or missense mutation), so long as the TRs mediate the desired function, such as viral packaging, integration, and / or proviral rescue. Typically, but not necessarily, the TRs are derived from the same parvovirus, e.g., both TR sequences are derived from AAV2.

[0123] The viral Rep proteins used to generate the double-stranded vectors are selected taking into account the origin of the viral TR, for example, AAV5 TR interacts more efficiently with AAV5 Rep proteins.

[0124] The genomic sequences of the different serotypes of various autonomous parvoviruses and AAV, as well as the sequences of the TR, capsid subunit, and Rep proteins, are known in the art. Such sequences can be found in the literature or in public databases such as GENBANK®. See, for example, GENBANK® Accession Nos. NC002077, NC001863, NC001862, NC001829, NC001729, NC001701, NC001510, NC001401, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC001358, NC001540, the disclosures of which are incorporated herein by reference in their entireties. See, e.g., Chiorini et al., (1999) J. Virology 73:1309; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, the disclosures of which are incorporated herein by reference in their entireties.

[0125] Non-releasable TRs can be made by any method known in the art. For example, an insertion into a TR replaces the nicking site (i.e., trs), resulting in a non-releasable TR. The nomenclature of various regions or elements within a TR is known in the art. A diagram of the regions within an AAV TR is provided in FIG. 6 (see also BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69, Figure 5, 3d ed., Lippincott-Raven Publishers). The insertion is preferably made in the sequence of the terminal releasable site (trs). Alternatively, the insertion may be made in the site between the Rep binding element (RBE) in the A element and the trs in the D element (see FIG. 6). The core sequence of the AAV trs site is known in the art and described in Snyder et al., (1990) Cell 60:105; Snyder et al., (1993) J. Virology 67:6096; Brister & Muzyczka, (2000) J. Virology 74:7762; Brister & Muzyczka, (1999) J. Virology 73:9325, the entire disclosures of which are incorporated herein by reference. For example, Brister & Muzyczka, (1999) J. Virology 73:9325 describes the 3'-CCGGT / TG-5' residue in the D element. *Snyder et al., (1993) J. Virology 67:6096, identified the minimal trs sequence as 3'-GGT / TGA-5', which is substantially overlapping with the sequence identified by Brister & Muzyczka. Preferably, the insertion is made in the region of the trs site. The insertion can be of any suitable length that reduces or substantially eliminates (e.g., 60%, 70%, 80%, 90%, 95% or more) TR dissociation. In some embodiments, the insertion is at least about 3, 4, 5, 6, 10, 15, 20, or 30 nucleotides or more. There is no particular upper limit to the size of the inserted sequence (e.g., the insertion can be as long as 50, 100, 200, or 500 nucleotides or more), as long as a suitable level of viral replication and packaging is achieved.

[0126] In another embodiment, the TR can be rendered non-dissociable by deletion of the trs site. The deletion can extend beyond the trs site by 1, 3, 5, 8, 10, 15, 20, 30, or more nucleotides, so long as the template retains the desired function. In addition to the trs site, part or all of the D element can be deleted. The deletion can extend further into the A element, but one of skill in the art will appreciate that it can be advantageous to retain the RBE within the A element, for example, to facilitate efficient packaging. The deletion within the A element can be 2, 3, 4, 5, 8, 10, or 15 nucleotides, or more, so long as the non-dissociable TR retains other desired functions. It is further preferred to delete part or all of the parvovirus (e.g., AAV) sequence beyond the D element outside the TR sequence (e.g., to the right of the D element in FIG. 6) to prevent gene conversion to correct the altered TR.

[0127] As a further alternative, the sequence of the nicking site can be mutated so that dissociation by Rep protein is reduced or substantially eliminated. For example, A and / or C bases can be substituted with G and / or T bases at or near the nicking site. The effect of substitutions at the terminal dissociation site on Rep cleavage is described by Brister & Muzyczka, (1999) J. Virology 73:9325, the disclosure of which is incorporated herein by reference. As a further alternative, nucleotide substitutions in the region surrounding the nicking site, which are hypothesized to form a stem-loop structure, can also be used to reduce Rep cleavage at the terminal dissociation site.

[0128] One of skill in the art will appreciate that modifications in the non-releasable TR can be selected to maintain the desired functions, if any, of the altered TR (e.g., packaging, Rep recognition, site-specific integration, etc.). In a more preferred embodiment, the TR will be resistant to the process of gene conversion as described in Samulski et al., (1983) Cell 33:135. Gene conversion in the non-releasable TR will restore the trs site, thereby generating a releasable TR and resulting in an increased frequency of monomeric replication products. Gene conversion occurs by homologous recombination between the releasable TR and the altered TR.

[0129] One strategy to reduce gene conversion is to use cell lines (preferably mammalian) known in the art that are defective in DNA repair to generate viruses, since these cell lines are impaired in their ability to correct mutations introduced into viral templates. Alternatively, templates with substantially reduced gene conversion rates can be generated by introducing heterologous regions into the non-releasable TR. Non-homology in the region surrounding the trs element between the non-releasable TR and the unmodified TR on the template will reduce or even substantially eliminate gene conversion.

[0130] Any suitable insertion or deletion can be introduced into the non-releasable TR to generate a heterologous region, as long as gene conversion is reduced or substantially eliminated. A strategy that utilizes deletion to create heterologous is preferred. It is further preferred that the deletion does not excessively impair template replication and packaging. In the case of deletion, the same deletion may be sufficient to impair the release of the trs site and reduce gene conversion. As a further alternative, gene conversion can be reduced by inserting into the non-releasable TR or into the A factor between the RBE and the trs site. The insertion is typically at least about 3, 4, 5, 6, 10, 15, 20, or 30 nucleotides in length, or more. There is no particular upper limit to the size of the inserted sequence, which may be 50, 100, 200, or 500 nucleotides, or more, but it is preferred that the insertion does not excessively impair template replication and packaging.

[0131] In another embodiment, the non-dissociable TR can be a naturally occurring TR (or a modified version thereof) that is non-dissociable under the conditions used. For example, the non-dissociable TR cannot be recognized by the Rep protein used to generate vDNA from the template. By way of example, the non-dissociable TR can be an autonomous parvovirus sequence that is not recognized by the AAV Rep protein. As a further alternative, the non-dissociable sequence can be an inverted repeat sequence that forms a hairpin structure and cannot be cleaved by the Rep protein. As yet another alternative, a template half the size of the genome can be used to generate parvovirus particles that retain double-stranded vDNA generated from a template half the size of the genome, as described in the examples herein and in Hirata & Russell, (2000) J. Virology 74:4612. This report describes the packaging of paired monomers and transient RF intermediates when the AAV genome is reduced to less than half the size (<2.5 kb) of the wtAAV genome. These researchers found that monomeric genomes were the preferred substrate for gene correction by homologous recombination, and that double-stranded genomes were less functional than monomeric genomes in this assay. This report did not explore or suggest the use of double-stranded genomes as vectors for gene delivery.

[0132] In some embodiments, the template will be approximately half the size of the vDNA that can be packaged by the parvovirus capsid. For example, in the case of an AAV capsid, the template is preferably approximately half the length of the wt AAV genome, as described above. The template (described above) is replicated to generate a double-stranded vector genome (vDNA) that can form double-stranded DNA under appropriate conditions. The double-stranded molecule is substantially self-complementary (i.e., at least 90%, 95%, 98%, 99% or more nucleotide sequence complementarity) so that double-stranded viral DNA can be formed. Base pairing between individual nucleotide bases or polynucleotide sequences is well understood in the art. Preferably, the double-stranded parvovirus viral DNA is essentially completely self-complementary (i.e., contains no or only an insignificant number of mismatched bases). In particular, it is preferred that the heterologous nucleotide sequence (e.g., the sequence transcribed by the cell) is essentially completely self-complementary.

[0133] In general, double-stranded parvoviruses may contain non-complementarity to the extent that the expression of heterologous nucleotide sequences from double-stranded parvovirus vectors is more efficient than the corresponding monomeric vectors.Double-stranded parvoviruses provide host cells with double-stranded molecules that address the host cell's need to convert single-stranded rAAV vDNA into double-stranded DNA.The presence of substantial non-complementary regions within virion DNA, particularly within the heterologous nucleotide sequence, will likely be recognized by host cells, resulting in the recruitment of DNA repair mechanisms to correct mismatched bases, thereby countering the advantageous features of double-stranded parvovirus vectors.For example, the vectors reduce or eliminate the need for host cells to process viral templates.

[0134] Lysosomal storage disorders and transgenes Disclosed herein is the surprising discovery of different expression patterns after intraventricular administration of scAAV9 and ssAAV9. In some embodiments, intraventricular administration of scAAV9 or ssAAV9 can be used to treat lysosomal storage disorders.

[0135] Lysosomal storage disorders are a type of disease that involves partial or complete deficiency of lysosomal hydrolases. This deficiency leads to incomplete lysosomal digestion of substrates specific to hydrolases. Over time, the accumulation of undigested substrates can lead to a variety of abnormalities, including progressive and severe neurodegeneration and muscular degeneration (Settembre et al., Human Mol.Genet., 17:119-129, 2008; Fukuda et al., Curr.Neurol.Neurosci.Rep., 7:71-77 2007).

[0136] Lysosomal protein defects generally result in deleterious accumulation of metabolites: mucopolysaccharide accumulation in Hurler, Hunter (mucopolysaccharidosis II), Morquio, and Sanfilippo syndromes; sphingolipid accumulation in Tay-Sachs, Gaucher, Krabbe, Niemann-Pick, and Fabry syndromes; and fucose-containing sphingolipids and glycoprotein fragments, as well as mannose-containing oligosaccharides, in fucosidoses and mannosidoses, respectively.

[0137] Enzyme replacement therapy (ERT) as a treatment for lysosomal storage diseases (LSDs) was proposed by De Duve and Wattiaux as early as 1966. However, it took more than 35 years to prove the safety and efficacy of ERT for lysosomal storage disorders (Gaucher disease type 1) (Charrow, Expert Opin. Biol. Ther.,9:121-31, 2009). The principles elaborated in the development of treatment for Gaucher disease were subsequently applied to the development of ERT for other LSDs. The safety and efficacy of ERT for Fabry disease (Zarate and Hopkin, Lancet, 18:1427-1435, 2008), mucopolysaccharidosis (MPS) I, MPS II, and MPS VI (Clarke, Expert Rev. Mol. Med., 10:e1, 2008), and Pompe disease (van der Beek, Acta Neurol. Belg., 106:82-86, 2006) have been demonstrated in well-designed clinical trials, and treatments are now commercially available (for reviews, see, e.g., Rohrbach and Clarke, Drugs, 67:2697-2716, 2007 and Burrow et al., Curr. Opin. Pediatr., 19:628-625, 2007). However, some manifestations of LSD do not respond to ERT treatment. Furthermore, the long-term effectiveness of most treatments has yet to be established.

[0138] Pompe disease (also known as glycogen storage disease type II (GSDII)) is a form of lysosomal storage disorder caused by a partial or complete deficiency of lysosomal acid alpha-glucosidase (GAA). GAA is responsible for the breakdown of glycogen in lysosomes, and the enzyme deficiency leads to the accumulation of glycogen primarily in skeletal and cardiac muscles. The disease is divided into two broad categories: infantile-onset and late-onset. Patients with the infantile form generally die within the first year of life due to cardiopulmonary failure. Late-onset forms occur any time after infancy and generally do not involve the heart, but a progressive skeletal myopathy leads to eventual respiratory failure. It is estimated that 1 in 40,000 individuals has some form of Pompe disease. For a review, see Fukuda et al., Curr.Neurol.Neurosci.Rep., 7:71-77 2007.

[0139] ERT treatment for Pompe disease involves intravenous injection of recombinant GAA (rhGAA) precursor containing mannose-6-phosphate (M6P) group. Genzyme Corporation sells commercial replacement enzymes under the trade names Myozyme® (injectable alglucosidase alfa) and Lumizyme®. The M6P group binds to the cation-independent mannose-6-phosphate receptor (CI-MPR) on the cell surface. The CI-MPR / rhGAA complex is internalized from the cell surface into a transport vesicle that fuses with the endosome. At the acidic pH of the late endosome, rhGAA dissociates from the CI-MPR and is transported to the lysosome, where it rescues the GAA defect. ERT for Pompe disease has been reviewed, for example, by Fukuda et al. (Curr.Neurol.Neurosci.Rep., 7:71-77, 2007).

[0140] ERT for Pompe disease is effective in glycogen clearance in cardiac muscle but not from skeletal muscle (Raben et al., Acta Myologica, 26:45-48, 2007).Similarly, in genetically modified mice lacking expression of GAA (a mouse model of Pompe disease), ERT is effective in removing glycogen from type I muscle fibers but not from type II muscle fibers, which predominate in skeletal muscle (Raben et al., Molecular Therapy, 11:48-56, 2005).

[0141] Fabry disease is an X-linked hereditary lysosomal storage disorder caused by deficiency of α-galactosidase A, which results in the accumulation of the neutral glycosphingolipid globotriosylceramide (Gb3) in the walls of small blood vessels, nerves, dorsal root ganglia, renal glomerular and tubular epithelial cells, and cardiac myocytes. It is a complex multisystem disorder clinically characterized by chronic pain and acroparesthesia, gastrointestinal disturbances, characteristic skin lesions (angiokeratoma), progressive renal damage, cardiomyopathy, and stroke. Enzyme replacement therapy with intravenous infusion of recombinant human α-galactosidase A consistently reduces plasma Gb3 levels and removes lysosomal inclusions from vascular endothelial cells. The effects of ERT on other tissues are less clear, suggesting that treatment must be initiated early in the course of the disease to be optimally effective or that some complications of the disease do not respond to intravenously delivered enzyme (for reviews see Clarke, Ann. Intern Med., 20:425-433, 2007 and Desnick, Ann. Intern Med., 138:338-346, 2003).

[0142] Gaucher disease is a genetic disorder caused by a deficiency in the activity of the enzyme glucocerebrosidase, which is found primarily in lysosomes. This results in the accumulation of glucocerebroside in the lysosomes of macrophages, primarily in the reticuloendothelial system. The consequences of this abnormal storage include visceral problems such as hepatomegaly, splenomegaly, anemia, and thrombocytopenia, which cause fatigue, discomfort, infection, bleeding, and bruising; pain (acute or chronic) and bone problems such as bone crisis and avascular necrosis; and other problems such as lung disease, growth failure, and delayed puberty. The severity and rate of progression of symptoms vary considerably from patient to patient, ranging from asymptomatic to severe with early death. Gaucher disease is classified into three subtypes according to clinical features. Type I can occur at any age and has primarily visceral symptoms without neurological effects. Type II causes severe progressive brain disease and death occurs during infancy. Type III occurs in childhood and has neurological and visceral symptoms. See Connock et al., Health Technology Assessment, 10:iii-136, 2004; and Beutler, PLoS Med., 1:e21, 2004.

[0143] Imiglucerase (commercially available as CEREZYME™ from Genzyme Corporation) is a recombinant enzyme modified to contain mannose to enhance cellular uptake and delivery to lysosomes. It is given intravenously to replace the missing enzyme and is approved for use in symptomatic Type I disease and to treat visceral symptoms of Type III disease. Intravenous CEREZYME® cannot cross the blood-brain barrier and is ineffective for neurological findings. However, a transgene encoding imiglucerase can be included in a scAAV9 or ssAAV9 vector and used in the disclosed methods.

[0144] Hurler syndrome, also known as mucopolysaccharidosis type I (MPS I), Hurler disease, or gargoyle facies, is a genetic disorder that results in the accumulation of mucopolysaccharides due to a deficiency in αL-iduronidase, an enzyme responsible for the breakdown of mucopolysaccharides in the lysosomes (Tolar and Orchard, Biologics., 2:743-751, 2008). Without this enzyme, there is a buildup of heparan sulfate and dermatan sulfate in the body. Symptoms appear during childhood and early death can occur due to organ damage. MPS I is classified into three subtypes based on the severity of symptoms. All three types are due to a lack or insufficient levels of the enzyme αL-iduronidase. MPS IH or Hurler syndrome is the most severe of the MPS I subtypes. The other two types are MPS IS or Scheie syndrome and MPS I HS or Hurler-Scheie syndrome. Recombinant alphaL-iduronidase (IDUA) is used in ERT for MPS I to reduce IDUA substrate accumulation in MPS I subjects (Tolar and Orchard, Biologics., 2:743-751, 2008). These transgenes can be included in scAAV9 or ssAAV9 vectors and used in the disclosed methods.

[0145] Hunter syndrome (mucopolysaccharidosis II) is a mucopolysaccharidosis (MPS), a family of inherited disorders of glycosaminoglycan (GAG) catabolism (Neufeld et al., The Metabolic and Molecular Bases of Inherited Disease. New York., NY:McGraw-Hill;3421-3452, 2001). Hunter syndrome is a rare X-linked disorder. Each MPS is caused by a deficiency in the activity of a distinct lysosomal enzyme required for the stepwise degradation of GAGs, dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate (Neufeld et al., The Metabolic and Molecular Bases of Inherited Disease. New York, NY:McGraw-Hill;3421-3452, 2001). In affected patients, undegraded or partially degraded GAGs accumulate in lysosomes and are excreted in excess in the urine (Dorfman et al., Proc Natl Acad Sci USA, 43:443-4462, 1957). It is the accumulation or storage of GAGs in lysosomes that contributes to the signs and symptoms of these disorders. MPS is chronic and progressive. The biochemical cause of Hunter syndrome is a deficiency in the activity of iduronate-2-sulfatase (I2S), a lysosomal enzyme that catalyzes the removal of sulfate from the 2-position of L-iduronic acid in dermatan sulfate and heparin sulfate (Bach et al., Proc. Natl. Acad. Sci. USA., 70:2134-2213, 1973; Neufeld et al., The Metabolic and Molecular Bases of Inherited Disease. New York, NY: McGraw-Hill; 3421-3452, 2001). Thus, this gene can be included in scAAV9 or ssAAV9 vectors and used in the disclosed methods.

[0146] Idursulfase (Elaprase, Shire Human Genetic Therapies, Inc, Cambridge, MA) is a recombinant human I2S made in a human cell line that has been approved in the United States and the European Union for the treatment of Hunter syndrome. Randomized, placebo-controlled, double-blind clinical trials have shown clinical efficacy in patients treated with idursulfase compared to patients treated with placebo. Patients treated with idursulfase show a statistically significant improvement rate compared to placebo. In addition, urinary GAG excretion and liver and spleen volumes were significantly reduced from baseline with both isursulfase dosing regimens. Idursulfase was generally well tolerated, and the majority of treatment-emergent adverse events were consistent with the natural history of untreated Hunter syndrome. Based on the greater clinical response in the once-weekly group compared to the EOW group, idursulfase was approved for the treatment of MPS II in both the United States and the European Union at a dose of 0.5 mg / kg once weekly (Muenzer et al., Genet. Med., 8:465-473, 2006).

[0147] Mucopolysaccharidosis IV (MPS IV; also known as Morquio syndrome) is an autosomal recessive lysosomal storage disorder involving the accumulation of keratan sulfate (Tomatsu et al., Hum. Mol. Genet., 17:815-824, 2007). Two types are recognized: type A is a deficiency of the enzyme N-acetylgalactosamine-6-sulfate sulfatase; type B is a deficiency of the enzyme β-galactosidase. Clinical features are similar in both types, but appear milder in type B. Onset is between 1 and 3 years of age. Neurological complications include compression of spinal nerves and nerve roots due to extreme progressive skeletal changes, especially in the ribs and chest; conductive and / or sensorineural hearing loss, and corneal opacification. Intelligence is normal unless hydrocephalus develops and is left untreated. Physical growth is slow and often stops between 4 and 8 years of age. Skeletal abnormalities include bell-shaped chest, flattened or curved spine, shortened long bones, and malformations of the hips, knees, ankles, and wrists. ERT with recombinant N-acetylgalactosamine-6-sulfate sulfatase has been used to treat a mouse model of MPSIV that lacks expression of N-acetylgalactosamine-6-sulfate sulfatase (Tomatsu et al., Hum.Mol.Genet., 17:815-824, 2007).

[0148] Any of these lysosomal storage disorders can be treated using the methods disclosed herein, which may include enzyme replacement therapy and ICV administration of ssAAV9 and / or scAAV9 vectors encoding therapeutic genes.

[0149] Non-lysosomal storage disorders and transgenes Non-lysosomal storage disorders that may be treated using the methods disclosed herein include spinal muscular atrophy (SMA) types 1, II, III, IV (SMN1 gene, UBA1 gene, DYNC1H1 gene, VAPB gene), X-linked myotubular myopathy (MTM1 gene), catecholamine-induced polymorphic ventricular tachycardia (CASQ2 gene), color vision disorders (CNGB3 gene, CNGA3 gene, GNAT2 gene, PDE6C gene), total choroidal atrophy (CHM gene), Friedreich's ataxia CNS (FXN gene), generalized Friedreich's ataxia CNS (FXN gene), and schizophrenia (SMA) (Schizophrenia). Reich's ataxia (FXN gene), adrenoleukodystrophy (ABCD1 gene), Alzheimer's disease (APP gene, PPARγ gene), amyotrophic lateral sclerosis (SOD1 gene), Angelman syndrome (UBE3A gene), ataxia-telangiectasia (ATM gene), Charcot-Marie-Tooth syndrome (PMP22 gene), Cockayne syndrome (ERCC6 gene, ERCC8 gene), hearing loss (GJB2 gene), Duchenne muscular dystrophy (DMD gene), epilepsy (SCN1A gene), fragile X syndrome (FMR1 gene), Huntington's disease (HTT gene), Lesch-Nyhan syndrome (HGPRT gene), Maple syrup urine disease (BCKDHA gene, BCKDHB gene, DBT gene), Menkes syndrome (ATP7A gene), myotonic dystrophy (DMPK), narcolepsy (HLA gene), neurofibromatosis (NF1 gene), Parkinson's disease (LRRK2 gene, PARK2 gene, PARK7 gene, PINK1 gene, SNCA gene), phenylketonuria (PAH gene), Prader-Willi syndrome These include, but are not limited to, von Hippel-Lindau syndrome (VHL gene), Williams syndrome (CLIP2 gene, ELN gene, GTF2I gene, GTF2IRD1 gene, LIMK1 gene), Wilson's disease (ATP7B gene), or Zellweger syndrome (PEX1 gene).

[0150] Immunosuppressants In some embodiments, the immunosuppressant is a biological response modifier such as KINERET® (anakinra), ENBREL® (etanercept), or REMICADE® (infliximab), a disease-modifying antirheumatic drug (DMARD) such as ARAVA® (leflunomide), a steroid such as prednisone or cortisone, an immunosuppressant such as mycophenolate mofetil (MMF), a macrolide inhibitor of IL-2 such as sirolimus (rapamycin) or tacrolimus, celecoxib, choline magnesium trisalicylate, diclofenac, diclofenac potassium, diclofenac serotonin ... nonsteroidal anti-inflammatory drugs (NSAIDs) such as Enac XR, diflunisal, etodolac, etodolac ER, fenoprofen, oral flurbiprofen, ibuprofen, indomethacin, indomethacin SR, indomethacin suppositories, ketoprofen, ketoprofen ER, meclofenamic acid, meloxicam, nabumetone, naproxen, naproxen CR, naproxen ER, oxaprozin, piroxicam, rofecoxib, salsalate, sulindac, or tolmetin sodium, or other products such as HYALGAN® (hyaluronic acid) or SYNVISC® (Hylan G-F20).

[0151] Immunosuppressive therapy can include treatment with prednisolone, mycophenolate mofetil (MMF), and sirolimus or tacrolimus.

[0152] In some embodiments, the immunosuppressant does not cross the blood-brain barrier. EXAMPLES

[0153] Gene therapy of the central nervous system can be a powerful treatment for a number of neurological disorders. Serotype 9-based adeno-associated viral vectors (AAV9) are strong candidates for delivering gene-based therapy throughout the brain and spinal cord when administered intravenously, intrathecally, intracisternally, and intracerebroventricularly (ICV). Previous studies of ICV-delivered self-complementary AAV9 were performed in neonatal mice with single-dose delivery. However, more information is needed regarding the dose-response relationship for transduction efficiency in adult animals before clinical trials can be considered. As disclosed below, three doses of self-complementary AAV9 were administered to adult rats. High levels of transduction were observed in the hippocampus, cerebellum, and cerebral cortex, with increased transduction with increasing dose. Both neurons and astrocytes were transduced. There was no evidence of astrocytosis at the doses tested. Preliminary results from pigs receiving ICV self-complementary AAV9 are also presented. The results of these dosing studies in large animal models provide evidence that this approach is clinically valid.

[0154] Example 1 material and method Vector: Self-complementary AAV9 (scAAV9) was utilized. The expression vector uses a proprietary mini-chicken β-actin promoter to drive expression of enhanced green fluorescent protein (GFP) cDNA. In rat studies, vector titers were 1.2×10 per ml. 13 Vector genome (vg). For pig studies, scAAV9 expressing GFP from the cytomegalovirus enhancer / chicken β-actin promoter was purchased from the vector core at Nationwide Children's Hospital. It was used at 1.34 × 10 13 Used at a concentration of vg / ml.

[0155] Animal Surgery: Male Sprague-Dawley rats were obtained from Charles River Labs (catalog number 400, weighing 276-300 g) and were acclimated to the animal facility for 2-7 days before surgery was performed. Rats were provided with ad libitum food (Labdiet 5001; Labdiet, St. Louis, MO) and tap water and were maintained on a 12:12 h light:dark cycle. Individual rats were randomly assigned to treatment groups as follows:

[0156] Rats were anesthetized and maintained with 2% isoflurane / 98% oxygen (0.6 L / min) throughout the procedure. The head was fixed in a stereotaxic frame using a nose cone and ear bars. The top of the head was shaved and sterilized with ethanol and betadine, and sterile drapes were applied around the surgical site. Lidocaine was administered subcutaneously, and buprinorphine (0.1 mg / kg) was administered to reduce postoperative pain. A burr hole was drilled unilaterally in the skull using a dental drill, 0.8 mm posterior to the bregma and 1.6 mm lateral to the midline. Vector was delivered using a Hamilton syringe with a 27-gauge needle attached to a micromanipulator. 3.1 ul (low dose, 3.7 × 10 10 vg;n=3), 15.5ul (medium dose, 1.9×10 11 vg; n = 2), and 77.5 ul (high dose, 9.3 × 10 11 Three doses were evaluated: 1.5 × 10 vg; n = 3). Additionally, to account for differences in delivery volume, the lowest dose of virus (3.7 × 10 vg) delivered in the highest volume (77.5 μl) was 10 A fourth cohort using 100-mL 1.5-HT 2.5 vg) was included (low dose / high volume; n=3). Vector was injected at a rate of approximately 25ul / min, controlled by the manual injection knob on the micromanipulator. The needle was left in place for 1 minute after injection and then slowly withdrawn. The skin was closed and the animals were kept warm during recovery. After 3 weeks, animals were euthanized and perfused with saline followed by 4% paraformaldehyde (PFA). Brain, spinal cord, and liver tissues were post-fixed overnight in 4% PFA and subsequently stored frozen in sucrose. Tissues were frozen on dry ice and stored at -80°C before sectioning.

[0157] Domestic pigs (30 kg) were obtained from Palmetto Research Swine (Reevesville, SC). Animals were sedated with 0.8 mg / kg acepromazine, 33 mg / kg ketamine, and 0.04 mg / kg atropine. Anesthesia was maintained using isoflurane. Pain was managed with a fetanyl patch (100 μg / hour for 3 days). The skin was incised paramedianly and dissection was performed to the occipital keel. A burr hole was drilled with a high-speed Midas Legend approximately 1 cm posterior and medial to the ipsilateral supraorbital foramen. The dura was punctured with an 18-gauge needle. A proximal ventriculoperitoneal shunt catheter was inserted approximately 2.5 cm into the brain in a posterior and slightly medial direction toward the ipsilateral anterior horn of the right lateral ventricle until it was sensed that it had penetrated the ventricle. The exact location of the needle was determined by observing the flow of cerebrospinal fluid (CSF) after the stylet was removed. The position was confirmed by contrast ventriculography using Omnipaque-300 contrast dye. The catheter was advanced an additional 1 cm and 3 ml of CSF was removed. A MINJ-PD pump (Tritech Research, Los Angeles, California) was used to deliver AAV9-GFP (1.34 × 10 in 20 mM Tris pH 8.0, 1 mM MgCl2, 200 mM NaCl + 0.001% Pluronic F68). 13 1.5-4.5 ml of 1000 mg / ml of 1000 mg ...

[0158] Histology: 40 μm coronal sections were cut on a Leica CM1950 cryostat. Primary antibodies for GFP (Millipore AB3080P, Billerica, MA), GFAP (Millipore MAB360), NeuN (Millipore MAB377), S100β (Millipore MAB079-1), Fox-2 (Abcam ab167282, Cambridge, MA), calbindin (Sigma C9848, Saint Louis, MO), and GAD67 (Millipore MAB5406) ​​were used at 1:500 for immunohistochemistry. Antigen retrieval for S100β and Fox-2 was performed using citrate buffer (10 mM sodium citrate, pH 6.0) at 52°C or 95°C for 20 min, respectively. For fluorescence microscopy, AlexaFluor® 488-conjugated goat anti-rabbit secondary antibodies (Life Technologies A11070, Grand Island, NY) were used to detect GFP, and AlexaFluor® 596-conjugated goat anti-mouse secondary antibodies (Life Technologies A11032) were used at a 1:500 dilution to detect cell type markers. GFP + For chromogenic detection of cells, a biotin-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch 111-065-144, West Grove, PA) was used at a dilution of 1:250, followed by a Vector ABC kit and HRP detection system (Vector, Burlingame, CA). Slides were imaged by a Nikon (Tokyo, Japan) Eclipse E400 microscope with fluorescence filters (UV-2E / C, B-2E / C, and G-2E / C) and a Nikon Digital Sight controller with a DS-Qi1Mc camera and a DS-Fi1 camera.

[0159] Quantification of transduction: JPEG images of the hippocampus, cerebral cortex, and cerebellum were taken using a 4x objective. The light source intensity and exposure time were kept constant. Images were imported into ImageJ (http: / / imagej.nih.gov_ij / ) for analysis. The four AAV treatment groups were blinded to the individual performing the analysis. For each image, areas outside the region of interest were first masked (not included in the analysis). For the hippocampus and cerebral cortex, brain areas adjacent to these areas were excluded from the analysis. For the cerebellum, areas of the image outside the molecular layer were excluded. The three color channels (red, green, and blue) were split into three separate 8-bit grayscale images. The image from the green channel was used to calculate the percentage of pixels in the image with values ​​between 0 and a pre-determined threshold. Since the histochemical stain is black, pixels within this value range should correspond to those covering the stained part of the section. To minimize detection of background staining, PBS-treated controls were used to empirically set the threshold for each region. The percentage of pixels in the region of interest (i.e., unmasked pixels) was similarly determined using the red channel. The percentage of positively stained pixels was determined by dividing the percentage of pixels covering the region of interest by the percentage of pixels that were positively stained for GFP by multiplying by 100. + The percentage of regions of interest where the mean was 0.01 was obtained. For the cerebral cortex and hippocampus, four images per animal were analyzed, taken equally from the left and right sides of the brain, to average the differences between the injected and uninjected sides. Similarly, six images of the cerebellum were analyzed for each animal (two right, two middle, two left). Differences between treatment groups were determined by two-tailed t-tests.

[0160] Example 2 Biodistribution of transduction To determine the dose that can be used clinically, two major limiting factors were considered: the viral titer and the volume that can be safely delivered in a reasonable time. Viral titers of clinical grade vectors are generally in the range of 10 13 ~10 14The range of vector genomes (vg) / ml is 100 mg / ml. We estimate that 5-10% of the CSF volume can be delivered in a single bolus ICV injection, which corresponds to approximately 8-16 ml in adults. Based on these estimates, the maximum adult ICV dose is approximately 100 mg / ml. 15 vg. Scaling down the dose based on rat CSF volume, this dose would be approximately 3×10 12 vg. This dose represents the maximum value under ideal conditions, so the highest dose tested in this study was one-third of this maximum.

[0161] Adult Sprague-Dawley rats received a unilateral injection of scAAV9-GFP into the lateral ventricle and were sacrificed 3 weeks later. 10 vg), 15.5ul (medium dose, 1.9×10 11 vg), and 77.5 ul (high dose, 9.3 × 10 11 Three doses of virus (3.7 × 10 vg) were evaluated. Additionally, to account for differences in delivery volume, the lowest dose of virus (3.7 × 10 vg) delivered in the highest volume (77.5 μl) was 10 A fourth cohort using 100% GFP (low dose / high volume) was included (low dose / high volume). Representative images of high dose treated rat brains stained for GFP are presented in Figure 1. Two types of distribution were evident at low magnification. In the cerebral cortex and cerebellum, cells were distributed homogeneously in these regions. Qualitative analysis showed similar GFP expression in the injected and uninjected hemispheres, except for the area adjacent to the injection tract, which showed high GFP expression. In contrast, limbic regions such as the hippocampus and striatum showed different transduction patterns between the two sides of the brain after unilateral injection into the lateral ventricle. Sections of the cervical spinal cord stained positive for GFP (Figure 1c), especially in the high dose group. However, there was little evidence of transduction of cell bodies in the ventral gray matter. Instead, the majority of the staining appeared to be axonal projections from neurons outside the spinal cord.

[0162] Both neurons and glial cells were transduced by ICV delivery. Because the vector was delivered to the lateral ventricle, we first looked for transduction in the choroid plexus and ependymal cell layer. Both structures contained abundant GFP. + Cells were present in the cerebellum (Fig. 2a). Neurons were also transduced. Staining for GFP and the neuronal nuclear marker NeuN co-labeled cells in both the cerebral cortex and the granular layer of the hippocampus (Fig. 2b). Neurons were transduced throughout the cerebral cortex, but GAD67-expressing GABAergic neurons were not transduced (Fig. 2f). In the cerebellum, Fox2, a nuclear marker that labels Purkinje cells, co-localized with GFP staining, indicating transduction of this cell type (Fig. 2c). These GFP + The cells also stained positive for calbindin, which is expressed by several types of neurons, including Purkinje cells (Figure 2d) (13). Many cells with astrocytic morphology were transduced. However, no colocalization of GFAP and GFP was observed, except in the vicinity of the injection tract (Figure 2e). However, S100β, another marker of astrocytes, was not detected in these GFP-expressing cells. + It colocalized with the cells, indicating astrocyte transduction (Fig. 2g).

[0163] Example 3 Transduction efficiency in the brain GFP + Areas were measured in three brain regions: the cerebral cortex, the hippocampus, and the cerebellum (Figure 3). Increasing the dose of scAAV9 generally increased transduction in all three brain regions. In the hippocampus, 25%-35% of the areas were GFP-positive. + In the cerebellum, GFP staining was predominantly outside the granular layer. + Transduced cells, predominantly Purkinje neurons, occupied 5% to 32% of the area of ​​the molecular layer. The cerebral cortex had the lowest level of transduction of the three regions quantified, with 1% to 10% of the area expressing GFP. + It was.

[0164] Example 4 Non-CNS transduction To determine whether scAAV9 escaped to the periphery after ICV injection, liver samples were stained for GFP. Very few GFP cells were identified (data not shown, 18 cm of tissue evaluated from mid-dose rats). 2 1 positive cell per 100 cells), suggesting that the amount of scAAV9 that escaped from the CNS was small.

[0165] Example 5 Astrocyte activation The introduction of foreign viruses and proteins can induce immune responses in the CNS, often resulting in astrogliosis. To look for the presence of activated astrocytes, we stained brain sections from two rats treated with PBS and two rats treated with high dose scAAV9 for GFAP. We evaluated eight sections per animal (four from the cerebellum, four including the hippocampus and cerebral cortex) taken from areas adjacent to those depicted in Figure 1. GFAP staining was evaluated at low magnification to look for gross changes in astrocyte activation, and at high magnification in the cerebellum, hippocampus, and cerebral cortex to look for local changes. No discernible changes in staining were observed in animals treated with AAV compared to animals treated with saline.

[0166] Example 7 ICV injection into adult pigs Three adult domestic pigs were given 2.01 × 10 13 ~6.03×10 13 Unilateral injections of scAAV9-GFP were given at doses ranging from 0.01 to 0.05 vg. After 3 weeks, the animals were euthanized. Results from all three pigs were similar. Representative sections are shown in Figure 4. As in rats, there is substantial transduction of the cerebral cortex, hippocampus, and cerebellum. Morphologically, these cells appear similar in appearance to those found in rats, suggesting transduction of both neurons, including Purkinje cells and astrocytes.

[0167] We extended these observations to a large animal model by utilizing a dose-escalation study of ICV scAAV9-GFP in rats to investigate vector biodistribution using a range of doses expected in future clinical trials. Unilateral ICV injection of scAAV9-GFP resulted in uniform transduction throughout the cerebral cortex and cerebellum, although substantial differences between the two hemispheres were observed in deeper structures such as the hippocampus (Figure 1).

[0168] Without wishing to be bound by theory, it is possible that the majority of virus that reaches the cerebellum and cerebral cortex may transduce by migrating through the ventricular system, into the subarachnoid space, and then into the tissue, whereas deeper structures are likely transduced by vectors that pass through the ependymal layer through the ventricle tract. Whatever the origin of this difference, these results suggest that bilateral ICV injections may be preferable in clinical situations. ICV-scAAV9-GFP injections in pigs mimic the transduction pattern observed in rats (Figures 1 and 4). These data suggest that the transduction pattern observed in rats extends to the larger mammalian brain.

[0169] ICV delivery of ssAAV9 vectors has been achieved in both mice and dogs. There are two notable differences in the transduction patterns observed in these species. In mice treated at P21, the transduction profile is predominantly neuronal, with almost no transduction of Purkinje neurons. Astrocytes are poorly transduced (7). An identical pattern of transduction is found in adult dogs treated with ICV-scAAV9 (12). In contrast, as disclosed herein, rats and pigs treated with ICV-scAAV9 have substantial transduction of astrocytes and neurons, and there is substantial transduction of cells of the cerebellum, particularly Purkinje neurons. Thus, the cell tropism and distribution patterns in this study are significantly different from previously published ICV studies in other species.

[0170] ICV delivery of AAV9, either scAAV9 or ssAAV9, has not yet been performed in primates. However, cisternal delivery of scAAV9-GFP has been evaluated in macaques (14). The authors of this study reported colocalization of GFP with neuronal and astrocytic markers, and the overall pattern of transduction in the cerebral cortex and cerebellum appears to be similar to that observed in rats and pigs. One notable difference between this macaque study and ours is that primates express S100β + Astrocytes plus GFAP + Although we also show transduction of astrocytes, our rodent studies show that S100β in the whole brain + We found primarily astrocyte transduction, with colocalization of GFP with GFAP only around the injection tract.

[0171] Differences in AAV transduction may result from differences in dose, delivery method, age, and species. In recent years, it has become evident that astrocytes are not a monolithic collection of cells. GFAP, a commonly used marker for astrocytes, is not immunohistochemically detectable in subpopulations of these cells (15). Furthermore, different astrocyte subpopulations respond to stimuli in different ways (16), and the diversity of astrocyte subtypes may vary between species (17). Without wishing to be bound by theory, the differences in transduction of primate and rat astrocytes may be explained by the preference of AAV9 for a specific subpopulation of astrocytes (the subset labeled by S100β) in rats. Alternatively, the transduced GFAP in primates may be due to the lack of apoptosis. + It is possible that astrocytes are not present in rats.

[0172] Due to their evolutionary lineage, it is unexpected that rats, pigs, and primates exhibit different transduction patterns for AAV9 (a mixture of neuronal and astrocytic transduction, high levels of cerebellar transduction) from the patterns exhibited by mice and dogs (neuronal transduction, poor cerebellar transduction). In examining previous studies in more detail, we identified one variable that correlates with the transduction patterns. Our study and the primate study (14) used scAAV9, whereas the mouse study (7) and the dog study (12) utilized ssAAV9. scAAV vectors are superior to ssAAV vectors because they bypass the need for second strand synthesis (18-22). This results in an increased number of cells transduced, faster transgene expression, and higher levels of transgene production. However, ssAAV and scAAV vectors in the same cell type exhibiting different cell tropism have not been reported previously. The differences observed above suggest that Purkinje neurons and astrocytes are less amenable to targeting by ssAAV9 vectors compared to scAAV9 vectors, representing a possible barrier for therapy using ssAAV to target these cell types.

[0173] Without wishing to be bound by theory, one explanation for this difference is that these cell types exhibit substantially lower efficiency of second strand synthesis compared to cortical neurons, reducing the probability that ssAAV vectors reach a transcriptionally active state. This may point to differences in DNA repair pathways between these cell types that may contribute to Purkinje cells being more susceptible to degeneration in certain diseases.

[0174] Delivery of a foreign protein, simply a marker or therapeutic gene (in the case of individuals with two null mutations), carries the risk of an immune response against that protein that could cause loss of the transduced cells. Astrocytes, in particular, can serve as antigen-presenting cells (23). Thus, the S100β seen after scAAV9 delivery +Transduction of astrocytes may be of concern. Indeed, Samaranch et al. observed behavioral deficits in primates 3 weeks after intrathecal delivery of AAV9 that correlated with loss of Purkinje neurons. Interestingly, this loss was transgene-dependent. Expression of the foreign protein GFP caused behavioral abnormalities, whereas expression of the self-recognition protein hAADC had little or no adverse effects (24). No overt behavioral changes were observed in the rats used in the disclosed study, and staining for GFAP did not reveal signs of astrogliosis. However, the highest dose we used was somewhat lower than that used in the primate study by Samaranch et al. Although both studies investigated delivery of the virus to the CSF, the delivery methods were also different (ICV vs. cisterna magna). In clinical situations, immunosuppression may be required (3).

[0175] Numerous disorders, such as, but not limited to, lysosomal storage diseases, are good candidates for ICV AAV9-mediated therapy. The majority of these disorders are due to the deficiency of a single acid hydrolase (25). These enzymes are essentially required in all cells, but can be secreted from transduced cells and taken up by nearby deficient cells in a process known as cross-correction (26). The widespread transduction we observed seems ideal for the treatment of this type of disorder. Indeed, ICV AAV9 treatment of mucopolysaccharidosis IIIB mice resulted in substantial improvement of biochemical and histological measures of disease, as well as an increase in lifespan to normal or near-normal levels (12). Given the high levels of Purkinje neuron transduction observed in our study, diseases affecting these cells, such as spinocerebellar ataxia, are clearly targets for ICV scAAV9-mediated gene therapy. This delivery route avoids direct injection into the parenchyma and should be safer (27, 28).

[0176] Example 8 References TIFF2025041933000002.tif38160TIFF2025041933000003.tif224160TIFF2025041933000004.tif224160TIFF2025041933000005.tif52157

[0177] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. administering to the human subject an effective amount of a self-complementary adeno-associated virus serotype 9 (scAAV9) encoding a non-secreted therapeutic protein.

1. A method for delivering a non-secreted therapeutic protein to the central nervous system (CNS) of a human subject in need thereof, comprising:

2. administering to the human subject an effective amount of a self-complementary adeno-associated virus serotype 9 (scAAV9) encoding a therapeutic protein.

1. A method for delivering a therapeutic protein to neurons, Purkinje neurons, and / or astrocytes of a human subject in need thereof, comprising: administering scAAV9 by injection into the cerebrospinal fluid (CSF) via an intraventricular, intrathecal cisternal, or intrathecal lumbar route.

3. 3. The method of claim 2, wherein the therapeutic protein is a non-secreted therapeutic protein.

4. The method of any one of claims 1 to 3, wherein the human subject is an infant.

5. Approximately 7.4×10 12 ~Approx. 1.86×10 14 The method of claim 4, comprising administering a full dose of scAAV9 to the subject.

6. The method of any one of claims 1 to 3, wherein the subject is an adult.

7. Approximately 2.59×10 13 ~Approx. 6.51×10 14 The method of claim 6, comprising administering a full dose of scAAV9 to the subject.

8. The method of any one of claims 1 to 7, wherein the genetic disorder is a lysosomal storage disease.

9. The method of any one of claims 1 to 8, comprising administering to the subject a therapeutically effective amount of an immunosuppressant.

10. The method of claim 9, wherein the immunosuppressant reduces the activity of cytotoxic T cells.

11. 11. The method of claim 9 or 10, wherein the immunosuppressant reduces encephalitis.

12. The method of any one of claims 9 to 11, wherein the immunosuppressant is a nonsteroidal anti-inflammatory agent.

13. 13. The method of any one of claims 1 to 12, wherein the method reduces the risk of encephalitis in a subject.

14. The method of any one of claims 1 to 13, wherein scAAV9 transduces cells in the brain.

15. The method of any one of claims 1 to 13, wherein the scAAV9 transduces cells in the hippocampus, cerebellum, and cerebral cortex.

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