Rational polyploid AAV virions that cross the blood-brain barrier and elicit reduced humoral responses

Abstract

The present invention relates to a substantially homogeneous population of rational polyploid adeno-associated virus (AAV) virions that cross the blood-brain barrier (BBB), the rational polyploids comprising VP3 viral structural proteins from any AAV serotype that crosses the BBB. In some embodiments, the rational polyploids cross the BBB upon systemic or intrathecal administration to a subject. In some embodiments, the rational polyploid AAV virions comprise at least one VP1 and / or VP2 viral structural protein in addition to the VP3 protein. In some embodiments, the VP3 capsid protein is from a non-human primate, and in some embodiments, the VP3 capsid protein is an AAV rhesus serotype.

Description

[Technical Field]

[0001] Cross-reference application This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 175,954, filed April 16, 2021, and U.S. Provisional Patent Application No. 63 / 180,414, filed April 27, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy, created on April 26, 2021, is named 046192-098020PL02_SL.txt and is 139,904 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to methods for producing a population of rational polyploid adeno-associated virus (AAV) particles, virions, and viral capsids with desired properties, virions, substantially homogeneous populations of such virions, methods for producing substantially homogeneous populations, and uses thereof. More specifically, the present invention relates to polyploid AAV virion particles comprising VP3 structural proteins from any AAV serotype that cross the blood-brain barrier (BBB), wherein the polyploid AAV virions cross the BBB and / or transduce cells of the BBB upon systemic or intrathecal administration. [Background technology]

[0004] Background of the Invention Central nervous system (CNS) diseases are some of the most difficult to treat because the blood-brain barrier (BBB) ​​almost completely restricts the passage of many therapeutic drugs into the CNS. Adeno-associated virus (AAV) vectors have been widely used to treat various central nervous system (CNS) diseases. Due to the presence of the blood-brain barrier (BBB), early attempts at AAV-based treatment of CNS diseases were primarily performed via intracranial injection. For example, in the treatment of central nervous system (CNS; i.e., brain and spinal cord) disorders, delivery of AAV-based therapeutics is complicated; direct administration generally requires invasive surgery, and when administered systemically, the blood-brain barrier prevents AAV-based therapeutics from reaching the CNS. Furthermore, high doses of AAV-based therapeutics are required to induce sufficient transduction of target CNS tissues, thus leading to increased risk of side effects and / or production difficulties given the high doses required. Although the peripheral nervous system (PNS; i.e., nerve tissues outside the brain and spinal cord) may be considered more accessible for therapeutic intervention, some PNS tissues, such as the dorsal root ganglion, remain difficult to target. The AAV serotype AAV9 has been extensively studied for its ability to cross the BBB and transduce astrocytes, but its efficiency is limited. For example, systemic injection of AAV9 has been evaluated in clinical trials for multiple CNS diseases. However, the development of systemic AAV injection for treating CNS diseases still involves many challenges, such as the BBB crossing efficiency of AAV, peripheral toxicity caused by the expression of genes delivered by AAV, and the immune barrier against AAV in the blood.

[0005] To date, 12 AAV serotypes and more than 100 variants have been identified, as well as animal and non-human primate AAV serotypes, including rhesus macaques (AAVrh) and chimpanzees. Different serotype capsids have different infectivity in tissues or cultured cells, which depends on primary receptors and co-receptors on the cell surface or their own intracellular trafficking pathways. Although the application of AAV vectors has been proven safe and demonstrated therapeutic efficacy, differences exist between serotypes, including different transduction and biodistribution profiles.

[0006] Some clinical trials have demonstrated high transgene expression in preclinical mouse studies but lacked similar transgene expression in human clinical trials. There have also been reports of distinct species-specific differences in transgene expression between mice, non-human primates, and humans, including potential capsid-specific cytotoxic T lymphocyte (CTL) responses that eradicate AAV-transduced hepatocytes, leading to treatment failure. Therefore, results from these clinical trials highlight the need to explore effective methods for enhancing AAV transduction without increasing vector capsid load. Additionally, the majority of people are naturally exposed to AAV. As a result, a large proportion of the population develops neutralizing antibodies (NABs) against certain serotypes of AAV in their blood and other body fluids.

[0007] The full range of immune responses to adeno-associated viruses has been evaluated, including innate immune responses, cytotoxic T cell (CTL) responses, and humoral responses. Pre-existing anti-AAV immunity, particularly neutralizing antibodies (NAb) against AAV serotypes, has emerged as a major challenge to the clinical application of AAV vector-mediated gene delivery. Several studies have discussed the prevalence of pre-existing NAb against commonly used AAV serotypes (e.g., AAV serotypes 1–9). Several studies have shown that the induction of antibodies due to natural exposure to AAV during childhood may impair the subsequent use of AAV as a gene therapy vector, and / or that the strong humoral responses induced by AAV vectors may impair the potential requirement for repeated administration of the same AAV vector (Hurlbut et al., Mol Ther, 2010, 18(11):1983; Manno et al., Nat Med, 2006, 12(3):342; and Wang et al., Blood, 2006, 107(5):1810; Calcedo et al., Front Immunol, 2013, 4, 341; the contents of each of which are incorporated herein by reference in their entirety). Therefore, humoral responses to AAV vectors pose a significant barrier to effective gene transfer, resulting in clearance of AAV vectors before they can enter target cells. Antibodies against the AAV capsid are highly prevalent in humans, the natural host of this virus, and cross-react with a wide range of serotypes depending on the degree of homology in the capsid protein sequence. Because NAbs can efficiently block AAV-mediated transduction in vivo, strategies to overcome humoral immunity against the viral capsid are crucial for achieving successful gene transfer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hurlbut et al., Mol Ther, 2010, 18(11):1983 [Non-patent document 2] Manno et al., Nat Med, 2006, 12(3):342 [Non-patent document 3] Wang et al., Blood, 2006, 107(5):1810 [Non-patent document 4] Calcedo et al., Front Immunol, 2013, 4, 341 Summary of the Invention [Means for solving the problem]

[0009] Therefore, there is a need to tailor AAV vectors that efficiently cross the BBB and have a complex set of desirable characteristics, including different transduction efficiencies and reduced immune responses, that would be highly beneficial in gene therapy drug development.

[0010] Summary of the Invention The technology herein relates to a substantially homogeneous population of rational polyploid or haploid AAV vectors, virions, or pharmaceutical compositions thereof that cross the blood-brain barrier (BBB) ​​and / or have reduced antigenicity. It is well known that gene therapy vectors and viral vectors deliver therapeutic genes to the CNS through the blood-brain barrier. The blood-brain barrier is a semi-permeable boundary of endothelial cells that prevents certain chemicals and molecules in the bloodstream from crossing and entering the extracellular fluid of the central nervous system. Herein, the inventors rationally designed AAV virions to contain structural VP proteins from more than one serotype to increase vector transduction efficiency into the CNS and PNS after systemic and intrathecal delivery. Embodiments of the present invention are based on the surprising discovery that the VP3 viral structural protein from an AAV serotype known to efficiently cross the blood-brain barrier is responsible for the blood-brain crossing phenotype. Rational polyploids can therefore be designed to cross the blood-brain barrier and avoid immune responses to the parent serotype, e.g., thereby evading neutralizing antibodies and eliciting less of a humoral response to the parent serotype from which they are created.

[0011] Previous reports have shown that the design of AAV vectors, such as rational polyploids, which are composed of capsids from two or more AAV serotypes, can utilize individual serotypes to modify properties such as tropism, transduction, or antigenicity. Some of these polyploid viruses have the ability to alter tropism and transduction efficiency, as well as the ability to evade neutralization by neutralizing antibodies (NABs).

[0012] The present inventors have previously discovered methodologies that enable the rational design and production of virions, sometimes referred to as rational polyploid virions, to indicate that the capsid proteins VP1, VP2, and VP3 are not all from the same serotype, but rather from at least two different serotypes. Sometimes the term haploid is used to refer to virions in which the capsid proteins VP1, VP2, and VP3 are from at least two different serotypes, and the term triploid is used to generally refer to virions in which the capsid proteins VP1, VP2, and VP3 are from three different serotypes. In particular, such rational polyploids, e.g., rational haploid virions, and methods for their production are disclosed in U.S. Patent No. 10,550,405, the entire contents of which are incorporated herein by reference.

[0013] The technology herein generally relates to a homogeneous population of rational polyploid adeno-associated virus (AAV) particles, virions, and viral capsids comprising a VP3 structural protein from any AAV serotype that crosses the blood-brain barrier (BBB), wherein the polyploid AAV virions cross the BBB upon systemic or intrathecal administration and / or transduce blood components to enable delivery to the brain via the cerebral circulation. In some embodiments, the VP3 capsid protein is from a non-human primate, such as the AAV rhesus monkey (rhAAV) serotype. The rational polyploid AAV virion may also comprise at least one VP1 and / or VP2 viral structural protein from a serotype different from the VP3 protein. In some embodiments, the rational polyploid AAV virion comprises a VP1 capsid protein from AAV8 and at least one VP3 capsid protein from any AAV serotype that crosses the BBB. In some embodiments, viral structural proteins (e.g., any one or more of VP1, VP2, or VP3) can be modified, for example, by altering chemically modified nucleotides, mixed serotypes, etc.

[0014] For example, the use of a VP3 structural protein from any AAV serotype that efficiently crosses the BBB alters the biodistribution and transduction efficiency of the vector after systemic or intrathecal administration, particularly demonstrating an increased ability of the AAV vector to cross the BBB and transduce one or more tissues in the CNS or peripheral nervous system (PNS). In some embodiments, the AAV polyploid virions disclosed herein exhibit altered biodistribution and an increased ability of the AAV vector to transduce brain vasculature (BBV) and / or blood components, e.g., cells in the blood, enabling delivery of AAV-transduced cells to the brain (or CNS or PNS) via cerebral circulation. In some embodiments, the rational polyploid vector comprises a VP3 viral structural protein from any serotype selected from the group consisting of AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43. In some embodiments, rational polyploid vectors comprise the VP3 viral structural protein from any non-primate AAV serotype, such as a rhesus AAV serotype.

[0015] In some embodiments, a rational polyploid vector comprises VP1 or VP2 structural proteins, or both VP1 and VP2 structural proteins, from a serotype that efficiently crosses the BBB, such as AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43. In other embodiments, a rational polyploid vector disclosed herein comprises VP1 or VP2 structural proteins, or both VP1 and VP2 structural proteins, from a serotype that does not cross the BBB. In some embodiments, a rational polyploid vector comprises VP1 or VP2 structural proteins, or both VP1 and VP2 structural proteins, from any non-primate AAV serotype, such as the rhesus AAV serotype (rhAAV, or AAV rh), provided that at least one of the structural proteins is different from the rhesus serotype. Alternatively, in some embodiments, a rational polyploid vector comprises VP1 or VP2, or both VP1 and VP, that is not from a non-primate AAV serotype. Non-limiting examples of AAV serotypes from which the VP3 of the rational polyploid populations of the present invention can be selected are described in PCT / US2018 / 066551 (WO2019126356A1), filed December 19, 2018, or PCT / US2014 / 055490 (WO2015038958), filed September 12, 2014, or Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021, each of which references is incorporated herein by reference in its entirety.Non-limiting examples of AAV rhesus serotypes from which VP3 of the rational polyploid populations of the present invention may be selected include AAVrhlO, AAV rh74, AAV rh39, AAV rh43, AAV rh38, AAV rh40, AAV rh2, AAV rh25, AAV rh57, AAV rh50, AAV rh49, AAV rh58, AAV rh61, AAV rh52, AAV rh53, AAV rh51, AAV rh64, AAV rh8, AAV rh1, AAV rh62, AAV rh48, AAV rh54, AAV rh55, AAV rh35, AAV rh37, AAV rh8, AAV rh10 ...62, AAV rh48, AAV rh54, AAV rh55, AAV rh35, AAV rh37, AAV rh8, AAV rh10, AAV rh10, AAV rh10, AAV rh10, AAV rh10, AAV rh10, AAV rh10, AAV rh10, AAV rh10, rh36, AAV rh13, AAV rh32, AAV rh33, and AAV rh34.

[0016] In some embodiments, the rational polyploid vectors disclosed herein exhibit enhanced binding to brain microvascular endothelial cells (BMVECs) compared to AAV8 vectors. In some embodiments, a population of rational polyploid AAV virions exhibits enhanced binding to brain microvascular endothelial cells (BMVECs) compared to AAV8, AAV9, PHP.B, or PHP.eB. In some embodiments, the population exhibits at least 2-fold enhanced binding, at least 3-fold enhanced binding, at least 4-fold enhanced binding, at least 5-fold enhanced binding, at least 6-fold enhanced binding, at least 7-fold enhanced binding, at least 8-fold enhanced binding, at least 9-fold enhanced binding, at least 10-fold or more enhanced binding compared to AAV8. In some embodiments, a population of rational polyploid AAV virions exhibits equivalent binding to BMVECs compared to AAV9, PHP.B, or PHP.eB. In some embodiments, a population of haploid AAV virions exhibits enhanced penetration into brain microvascular endothelial cells (BMVECs) compared to AAVs that do not efficiently cross the blood-brain barrier, such as AAV 8, AAV 2, or AAV 5. In some embodiments, the rational polyploid vectors disclosed herein exhibit enhanced transduction of one or more of the cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord of a subject compared to non-rational polyploid AAV particles that lack the ability to efficiently cross the blood-brain barrier. In some embodiments, the rational polyploid vectors disclosed herein exhibit enhanced transduction compared to any one of AAV2, AAV8, or AAV5 in one or more CNS regions selected from the group consisting of the medullary plexus, cervical plexus, thoracic plexus, lumbar plexus, and choroid plexus.

[0017] In some embodiments, the rational polyploid vectors disclosed herein are used to transduce cells or tissues of the CNS. CNS cells can be, but are not limited to, neurons (e.g., excitatory neurons, inhibitory neurons, motor neurons, sensory neurons, autonomic neurons, sympathetic neurons, parasympathetic neurons, Purkinje neurons, Betz neurons, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes), and / or brain support cells, such as immune cells (e.g., T cells). CNS tissues can be, but are not limited to, the cortex (e.g., frontal cortex, parietal cortex, occipital cortex, temporal cortex), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei. In some embodiments, the rational polyploid vectors disclosed herein are used to transduce cells or tissues of the PNS. PNS cells or tissues can be, but are not limited to, dorsal root ganglia (DRG).

[0018] In some embodiments, the rational polyploid vectors disclosed herein have a biodistribution in the CNS that, in some embodiments, is the same (i.e., equivalent) or greater (i.e., increased) than the biodistribution of AAV9 in the CNS.

[0019] In some embodiments, the rational polyploid vectors disclosed herein have a vg / cell value of at least 0.05 vg / cell, 0.1 vg / cell, at least 0.2 vg / cell, at least 0.4 vg / cell, at least 0.6 vg / cell, at least 0.8 vg / cell, at least 1 vg / cell, at least 5 vg / cell, at least 10 vg / cell, at least 20 vg / cell, at least 25 vg / cell, or preferably greater.

[0020] In some embodiments, the rational polyploid vectors disclosed herein elicit a reduced or lower humoral immune response compared to the humoral response elicited by the parent AAV VP1 or AAV VP2 serotype—i.e., for example, if the rational polyploid vector comprises VP1 and / or VP2 from an AAV8 serotype and VP3 from a serotype that crosses the BBB, the humoral response elicited by the rational polyploid vector is reduced compared to the AAV8 parent virion. The reduction in humoral response can be at least 10%, 20%, 30%, 40%, or greater than 40% compared to the parent AAV VP1 or AAV VP2 serotype. In one embodiment, the humoral response is reduced compared to the humoral response elicited by the parent AAV VP3 serotype. In some embodiments, the rational polyploid vectors disclosed herein elicit a reduced or lower humoral immune response compared to the humoral response elicited by the parent AAV VP3 serotype—i.e., for example, when a rational polyploid vector comprises VP1 and / or VP2 from the AAV8 serotype and a VP3 from an AAVrh serotype that crosses the BBB, such as an AAVrh serotype such as AAVrh10 or AAVrh74, the humoral response elicited by the rational polyploid vector is reduced compared to the AAVrh10 or AAVrh74 parent virion. In some embodiments, the reduction in humoral response can be at least 10%, 20%, 30%, 40%, or greater than 40% compared to the parent AAV VP3 serotype. In certain aspects of the embodiments, the VP3 is selected from a non-human parvovirus serotype, e.g., an AAVrh such as AAVrh10 or AAVrh74 disclosed herein and in the Examples. In some embodiments, due to the reduced humoral immune response compared to the humoral immune response to the parent serotype, it allows for repeated dosing, e.g., the rational polyploid vectors disclosed herein can be administered multiple times, e.g., an initial dose followed by one or more subsequent doses (e.g., boosters).

[0021] In some embodiments, the rational polyploid vectors disclosed herein evade neutralizing antibodies against the parent serotypes of the VP1, VP2, or VP3 viral structural proteins—i.e., for example, if the rational polyploid vector comprises VP1 and / or VP2 from the AAV8 serotype and VP3 from a serotype that crosses the BBB, such as the AAV9 serotype, the rational polyploid vector evades neutralizing antibodies against the parent AAV8 and / or AAV9 serotypes. In some embodiments, the amount of neutralization of the rational polyploid vector from anti-AAV neutralizing antibodies against the parent serotype is less than 30%, or less than 20%, or less than 10%, or even less than 10%. Simply for illustration, in some embodiments, if anti-AAV antibodies against the parent serotype neutralize the parent AAV serotype by 50%, then the anti-AAV antibodies against the parent serotype will neutralize or inactivate the rational polyploid by 40%, or less than 30%, or less than 20%, or less than 10%, or less than 10%.

[0022] In some embodiments, disclosed herein relates to a population of rational polyploid AAV virions that allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and an AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype; the population of rational polyploid AAV virions elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and the VP1 and VP2 are not from the Rhesus AAV serotype, and the repeated dosing comprises a first administration of the population of rational polyploid AAV virions and a second administration of the parent AAV serotype of the VP1 structural viral protein or the VP2 structural viral protein.

[0023] Because rational polyploid virions have a reduced humoral immune response, the rational polyploid virions described herein allow for repeated dosing with a parent AAV serotype, for example, a repeated dosing comprising a first administration of the rational polyploid virion and a second administration of the parent AAV serotype used to provide the VP1 or VP2 structural protein of the rational polyploid virion. Simply for illustrative purposes, an AAV8-8-rh10 rational polyploid vector may be administered as the first dose, and the second dose may be an AAV8-8-8 serotype. In some embodiments, rational polyploid virions allow for repeated dosing, comprising a first administration of the rational polyploid virion and a second administration of the parent AAV serotype VP3 viral structural protein, where VP3 is from the AAV rhesus serotype. Solely for illustrative purposes, the AAV8-8-rh10 rational polyploid vector may be administered as the first dose and the second dose may be of the AAVrh10-rh10-rh10 serotype.

[0024] In some embodiments, at least one of the viral capsid proteins is a modified viral capsid protein. In some embodiments, at least one of the viral capsid proteins is a chimeric viral capsid protein. The viral capsid protein may be modified by substitution, insertion, or deletion of one or more amino acids. In some embodiments, at least one of the VP capsid viral proteins is not chimeric. In some embodiments, VP1 is a chimeric VP1 protein. In some embodiments, VP1 and VP2 are chimeric, and only VP3 is non-chimeric. For example, only viral particles are composed of VP1 / VP2 from a chimeric AAV2 / 8 (AAV2 N-terminus and AAV8 C-terminus) paired with only VP3 from any other non-AAV8 vector, such as rh10, rh74, etc. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. In another example, there are no chimeric ones.

[0025] In one embodiment, the AAV rational polyploid virions described herein, which encapsidate the AAV genome (including the heterologous gene located between two AAV ITRs), can be formed with only two of the viral structural proteins, VP1 and VP3. In one embodiment, such AAV rational polyploid virions are conformationally correct, i.e., have icosahedral symmetry with T=1. In one embodiment, the AAV haploid virions described herein are infectious. The ITRs can be from any serotype, e.g., AAV8 or AAV2, or from any of the 12 serotypes of AAV isolated for gene therapy, other species, mutant serotypes, shuffled serotypes of such genes, e.g., AAV1, AAV2, VP1.5, AAV4 VP2, AAV4 VP3, Rh10 VP3, Rh74 VP3, Rh74 VP2, or any other AAV serotype desired, e.g., as disclosed in Table 1.

[0026] In some embodiments, the substantially pure population of AAV rational polyploid virions disclosed herein comprises at least 10 1 virions, at least 10 2 virions, at least 10 3 virions, at least 10 4 virions, at least 10 5 virions, at least 10 6 virions, at least 10 7 virions, at least 10 8 virions, at least 10 9 virions, at least 10 10 virions, at least 10 11 virions, at least 10 12 virions, at least 10 15 virions, at least 10 17 In one embodiment, the population is at least 100 virus particles. In one embodiment, the population of AAV rational polyploid virions disclosed herein is at least 10 9 ~10 12 virions.

[0027] In one embodiment, the population is at least 1 x 10 4 viral genomes (vg) / ml or at least 1 × 10 5 viral genomes (vg) / ml or at least 1 × 10 6 viral genomes (vg) / ml, at least 1 × 10 7 viral genomes (vg) / ml, at least 1 × 10 8 viral genomes (vg) / ml, at least 1 × 10 9 Viral genomes (vg) / ml, at least 1 × 10 per ml 10 vg, at least 1 × 10 per ml 11 vg, at least 1 × 10 per ml 12 In one embodiment, the population is about 1 x 10 5 vg / ml ~ approx. 1×1013 vg / ml range.

[0028] In some embodiments of the polyploid AAV vectors disclosed herein useful in methods for treating diseases or disorders of the brain or spinal cord, or neuronal or neurodegenerative diseases, exemplary doses for therapeutic effect are titers of at least about 1.0E12 to 4.0E12 vg / kg, or about 1.2E12 to 3.0E12 vg / kg, or about 1.2E12 to 2.5E12 vg / kg, or about 2.5E12 to 4.0E12 vg / kg.

[0029] A substantially homogeneous population is at least 90%, at least 91%, at least 93%, at least 95%, at least 97%, at least 99%, at least 99.5%, or at least 99.9% of the desired AAV rational polyploids described herein. In one embodiment, the population is completely homogeneous. Accordingly, some aspects of the technology described herein relate to a system for producing substantially homogeneous haploid or rational polyploid AAV virions, the system comprising a vector containing a nucleic acid encoding VP1 from only one AAV serotype selected from Table 1, and optionally VP2 from only one AAV serotype selected from Table 1, and VP3 from any AAV serotype that efficiently crosses the BBB, such as AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43. In some embodiments, the VP3 protein is from a non-human primate, e.g., chimpanzee or rhesus AAV (AAVrh) serotype disclosed herein, e.g., AAVrh.10, AAVrh.74, AAVrh.73, AAVrh.75, AAVrh.76, rAAVrh.39, rAAVrh.43. In some embodiments, an exemplary system includes (i) a promoter operably linked to a nucleic acid encoding VP1 and VP2 from only a first AAV serotype but not expressing VP3 from the first AAV serotype, wherein the first AAV serotype is selected from any of the serotypes listed in Table 1, and (ii) a promoter operably linked to a nucleic acid encoding a VP3 protein from any AAV serotype that efficiently crosses the BBB as disclosed herein, e.g., AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43.

[0030] One embodiment provided herein is a population of rational polyploid AAV virions suitable for use in crossing the blood-brain barrier, wherein the rational polyploid AAV virions comprise at least one of AAV VP1 or VP2 viral structural proteins and an AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV serotype, and the VP3 viral structural protein is from an AAV serotype that efficiently crosses the blood-brain barrier and is different from at least one of the VP1 or VP2 serotypes; and the population of rational polyploid AAV virions crosses the blood-brain barrier (BBB) ​​and / or transduces endothelial cells of the BBB and / or blood components that cross the BBB upon systemic or intrathecal administration.

[0031] In one embodiment of any aspect herein, the population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​relative to non-rational polyploid AAV particles that lack the ability to cross the blood-brain barrier.

[0032] In one embodiment of any aspect herein, the VP3 viral structural protein is an AAV rhesus serotype.

[0033] In one embodiment of any aspect herein, the VP3 viral structural protein is from a serotype that efficiently crosses the blood-brain barrier selected from the group consisting of AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrh10, AAVrh74, AAVrh.39, and AAVrh43.

[0034] In one embodiment of any aspect herein, the population exhibits enhanced transduction of one or more of the cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord of a subject relative to non-rational polyploid AAV particles that lack the ability to efficiently cross the blood-brain barrier.

[0035] In one embodiment of any aspect herein, the rational polyploid AAV exhibits enhanced transduction compared to AAV2 in one or more CNS regions selected from the group consisting of the medullary plexus, cervical plexus, thoracic plexus, lumbar plexus, and choroid plexus.

[0036] In one embodiment of any aspect herein, the rational polyploid AAV exhibits enhanced binding to brain microvascular endothelial cells (BMVECs) compared to AAV8.

[0037] In one embodiment of any aspect herein, the population has a biodistribution in the CNS.

[0038] In one embodiment of any aspect herein, the CNS biodistribution is at least 0.05 vg / cell, 0.1 vg / cell, at least 0.2 vg / cell, at least 0.4 vg / cell, at least 0.6 vg / cell, at least 0.8 vg / cell, at least 1 vg / cell, at least 5 vg / cell, at least 10 vg / cell, at least 20 vg / cell, at least 25 vg / cell, or preferably more.

[0039] In one embodiment of any aspect herein, either VP1 or VP2 is selected from AAV serotypes that cross the blood-brain barrier.

[0040] In one embodiment of any aspect herein, either VP1 or VP2 is selected from AAV serotypes that do not cross the blood-brain barrier.

[0041] In one embodiment of any aspect herein, the VP1 or VP2 is not selected from an AAV rhesus serotype.

[0042] In one embodiment of any aspect herein, either VP1 or VP2 is selected from the AAV rhesus serotype.

[0043] In one embodiment of any aspect herein, the population elicits a humoral immune response that is reduced compared to the humoral response elicited by the parent AAV VP1 or AAV VP2 serotype.

[0044] In one embodiment of any aspect herein, the population escapes neutralizing antibodies against the parent serotype of the AAV VP1, VP2, or VP3 viral structural protein.

[0045] One aspect provided herein provides a method for delivering a transgene across the blood-brain barrier, the method comprising administering a population of any of the rational polyploid AAV virions described herein.

[0046] One embodiment provided herein provides a method for repeat doing comprising first and second administrations, wherein the repeat doing comprises a first administration of any of the rational polyploid AAV virions described herein and a second administration of a parent AAV serotype of a VP1 or VP2 viral structural protein, wherein the population of rational polyploid AAV virions elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and wherein VP1 or VP2 is not from the Rhesus AAV serotype.

[0047] One embodiment provided herein provides a population of rational polyploid AAV virions that allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype; the population of rational polyploid AAV virions elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and the VP1 and VP2 are not from the Rhesus AAV serotype, and the repeated dosing comprises a first administration of the population of rational polyploid AAV virions and a second administration of the parent AAV serotype of the VP1 structural viral protein or the VP2 structural viral protein.

[0048] One embodiment provided herein is a population of rational polyploid AAV virions, comprising: (a) AAV viral structural proteins VP1 and VP2 selected from an AAV8 viral serotype; and (b) a VP3 selected from an AAV rhesus serotype, AAV rh10, or AAVrh74;

[0049] A rational population of polyploid AAV virions is provided that elicits a humoral response that is reduced compared to that elicited by the parental AAV8 serotype.

[0050] One embodiment provided herein provides a method for repeated dosing, comprising a first and a second administration, wherein the first administration is a population of any of the rational polyploid AAV virions described herein, and the second administration is of a parent AAV serotype of a VP1 or VP2 viral structural protein, wherein the first administration elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and wherein VP1 or VP2 is not from a Rhesus AAV serotype.

[0051] In one embodiment of any aspect herein, the population escapes neutralizing antibodies against the parent serotype of the AAV VP1, VP2, or VP3 viral structural protein.

[0052] One aspect provided herein provides a method for delivering a transgene across the blood-brain barrier, the method comprising administering a population of any of the rational polyploid AAV virions described herein.

[0053] In one embodiment of any aspect herein, the VP3 protein is a mutant VP3 protein from the AAVrh10 or AAVrh74 serotype.

[0054] In one embodiment of any aspect herein, the mutant AAVrh74 VP3 protein has the amino acid sequence of SEQ ID NO:2, or the amino acid sequence of a protein having at least 85% sequence identity to SEQ ID NO:2, or the mutant AAVrh74 VP3 includes at least one of the following modifications of SEQ ID NO:2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P.

[0055] In one embodiment of any aspect herein, the mutant AAVrh10 VP3 protein is encoded by a nucleic acid of SEQ ID NO: 5 that includes at least one or more of Q214N, S462N, and D517E mutations compared to the AAVrh10_VP3 nucleic acid of SEQ ID NO: 5, or is encoded by a nucleic acid of SEQ ID NO: 5 that includes a nucleic acid sequence with at least 85% sequence identity to SEQ ID NO: 5 that includes at least one mutation selected from Q214N, S462N, and D517E.

[0056] In one embodiment of any aspect herein, the VP3 protein is an AAVrh74 VP3 protein comprising the amino acid sequence of SEQ ID NO:2 or 3, or the amino acid sequence of SEQ ID NO:2 or a protein having at least 85% sequence identity to SEQ ID NO:2, or the VP3 protein comprises at least one of the following amino acid modifications of SEQ ID NO:2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P.

[0057] One embodiment provided herein is a substantially homogenous population of any of the virions described herein, comprising at least 10 1 A population of individual virions is provided.

[0058] One embodiment provided herein provides a nucleic acid comprising, in a 5' to 3' direction: (a) a first nucleic acid encoding an AAVrh10 VP3 capsid protein operably linked to a first promoter; (b) a first polyA sequence; (c) a second nucleic acid encoding a rep protein; (d) a third nucleic acid encoding AAV8 VP1 and VP2 viral structural proteins, wherein the third nucleic acid is not capable of expressing the AAV8 VP3 viral structural protein; and (e) a second polyA sequence.

[0059] One embodiment provided herein provides a nucleic acid comprising, in a 5' to 3' direction: (a) a first nucleic acid encoding an AAVrh74 VP3 capsid protein operably linked to a first promoter; (b) a first polyA sequence; (c) a second nucleic acid encoding a rep protein; (d) a third nucleic acid encoding AAV8 VP1 and VP2 viral structural proteins, wherein the third nucleic acid is not capable of expressing the AAV8 VP3 viral structural protein; and (e) a second polyA sequence.

[0060] One embodiment provided herein provides a viral vector comprising: (a) any of the AAV virions described herein; and (b) a nucleic acid comprising at least one long terminal repeat sequence and a heterologous gene, wherein the nucleic acid is encapsulated in the AAV capsid.

[0061] In one embodiment of any aspect herein, the population comprises chimeric or modified viral structural proteins, wherein the modified viral structural proteins comprise one or more amino acid insertions, deletions, or substitutions.

[0062] In one embodiment of any aspect herein, the substantially homogeneous population produces significantly less anti-AAV IgG antibodies against the parent AAV serotype of VP1 or VP2 structural proteins in serum in vivo compared to a substantially homogeneous population of virions comprising the parent AAV serotype.

[0063] In one embodiment of any aspect herein, the parent AAV serotype is AAV8.

[0064] One embodiment provided herein provides a population of rational polyploid AAV virions that allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and an AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype; the population of rational polyploid AAV virions evades neutralizing antibodies against the parent AAV rhesus serotype of the VP3 viral structural protein, and VP1 and VP2 are not from the Rhesus AAV serotype; the repeated dosing comprises a first administration of the population of the parent AAV rhesus serotype of the VP3 structural protein and a second administration of the population of rational polyploid AAV virions, and the VP3 structural protein of the rational polyploid virions is the AAV rhesus mutant viral structural protein VP3.

[0065] In one embodiment of any aspect herein, the mutant AAV rhesus viral structural protein VP3 is from a mutant AAV rh10 VP3 viral structural protein or from a mutant AAV rh74 VP3 viral structural protein.

[0066] In one embodiment of any aspect herein, the mutant viral structural protein VP3 comprises mutations at amino acids corresponding to amino acids selected from the group consisting of N263, G264, T265, S26T, G268, T270, T274, E533, where all of the amino acid positions correspond to the native VP1 sequence numbering of AAV rh10 or AAVrh74.

[0067] In one embodiment of any aspect herein, the mutation is selected from the group consisting of N263S, G264A, T265S, S266T, G268A, T270del, T274H, and E533K.

[0068] In one embodiment of any aspect herein, the mutant viral structural protein VP3 further comprises a mutation at an amino acid corresponding to an amino acid selected from the group consisting of R727 and N737, where all of the amino acid positions correspond to the native VP1 sequence numbering of AAVrh10.

[0069] In one embodiment of any aspect herein, the mutation is selected from the group consisting of R727H and N737P.

[0070] In one embodiment of any aspect herein, the mutant viral structural protein VP3 further comprises a mutation at an amino acid corresponding to an amino acid selected from the group consisting of R726 and N736, where all of the amino acid positions correspond to the native VP1 sequence numbering of AAV rh74.

[0071] In one embodiment of any aspect herein, the mutation is selected from the group consisting of R726H and N736P.

[0072] In one embodiment of any aspect herein, the mutant viral structural protein VP3 further comprises a mutation at the amino acid corresponding to W at 581, where W is replaced with two subsequent V residues (VV), where all of the amino acid positions correspond to the native VP1 sequence numbering of AAV rh74.

[0073] In one embodiment of any aspect herein, the AAV VP1 or VP2 viral structural protein is any AAV serotype selected from Table 1.

[0074] In one embodiment of any aspect herein, the AAV VP1 or VP2 viral structural protein is AAV8.

[0075] In an additional embodiment, the present invention provides an AAV rational polyploid viral vector comprising (a) a rational polyploid AAV vector disclosed herein and (b) a nucleic acid comprising at least one long terminal repeat sequence, wherein the nucleic acid is encapsidated by the AAV rational polyploid virus. The AAV rational polyploid viral vector may be an AAV haploid particle, and the AAV rational polyploid viral vector proteins, capsid, viral vector and / or AAV haploid particle disclosed herein may be present in a composition further comprising a pharmaceutically acceptable carrier.

[0076] In a further embodiment, the invention provides a method of administering a nucleic acid to a cell, the method comprising contacting the cell with an AAV haploid viral vector of the invention and / or a composition of the invention.

[0077] Also provided herein are methods for delivering nucleic acid to a subject, the methods comprising administering to a subject the AAV haploid viral vectors and / or compositions of the invention.

[0078] Additionally, provided herein are AAV8 haploid capsid proteins, capsids, viral vectors, AAV particles and / or compositions of the invention for pharmaceutical use in the beneficial treatment of a disorder or disease.

[0079] In a further aspect, the present invention provides a population of rational polyploid AAV virions that allows for repeated dosing (i.e., an initial dose and one, or two, or three, or four, or five, or more than five subsequent doses or boosters), comprising at least one of AAV VP1 or VP2 viral structural proteins and an AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype (rhAAV), such that the population of rational polyploid AAV virions evades neutralizing antibodies against the parent AAV rhesus serotype of the VP3 viral structural protein, and VP1 and VP2 are selected from the rhesus AAV serotype. The population is not from an AAV serotype, and the repeated administration comprises a first administration of a population of VP3 structural proteins of a parent AAV rhesus serotype, and a second administration of a population of rational polyploid AAV virions, wherein the VP3 structural proteins of the rational polyploid virions are AAV rhesus mutant virus structural proteins. In some embodiments, the VP3 structural protein is a modified VP3 protein from rhAAV, for example, a modified VP3 protein from rh10 or rh74. In some embodiments, the modified VP3 protein is the rh10-LP2 VP3 protein disclosed herein. In some embodiments, the modified VP3 protein is the rh74-LP2 VP3 protein disclosed herein.

[0080] In some embodiments, the population of rational polyploid virions that allows repeated dosing comprises a first administration of rational polyploid AAV virions and a second administration of a parent AAV VP3 viral structural protein of a rhesus serotype, wherein the VP3 structural protein of the rational polyploid virions is a mutant AAV rhesus viral structural protein VP3, the population comprising at least one of AAV VP1 or VP2 viral structural protein and the AAV VP3 viral structural protein, wherein the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype, the population of rational polyploid AAV virions evading neutralizing antibodies against the parent AAV rhesus serotype of the VP3 viral structural protein, and at least one of VP1 and VP2 is not from the Rhesus AAV serotype. In some embodiments, both the VP1 structural protein and the VP2 structural protein are not from the Rhesus AAV serotype.

[0081] Rational polyploids comprising rhesus-modified VP3 proteins (e.g., rh10-VP3 proteins containing any one or more of the modifications disclosed herein: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R727H, N737P, or rh74-VP3 proteins containing any one or more of N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P) that are capable of evading neutralizing Abs against the parent AAV rhesus serotype. In some embodiments, the populations are capable of evading neutralizing Abs against the parent AAV VP1 or VP2 serotype, and VP1 or VP2 are not from a rhesus serotype.

[0082] These and other aspects of the present invention are addressed in more detail in the description of the invention set forth below.

[0083] This patent application contains at least one drawing executed in color. Copies of this application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. The accompanying drawings illustrate aspects of the invention.

[0084] The following detailed description, given by way of example and not intended to limit the invention to the particular embodiments described, can be understood in conjunction with the accompanying drawings, which are incorporated herein by reference: Various preferred features and embodiments of the present invention will now be described, by way of non-limiting examples, and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0085] [Figure 1-1] Figures 1A-1B show schematic diagrams of all-in-one constructs for generating AAV8 haploids disclosed herein. Figure 1A shows a schematic diagram of a construct for generating an AAV8-8-rh10 or AAV8-8-rh74 vector, comprising (i) a nucleic acid encoding the VP3 protein from the AAVrh10 or AAVrh74 serotype, followed by a promoter operably linked to a polyA sequence, (ii) a nucleic acid sequence encoding the Rep 2 gene (e.g., including promoters p5, p15, and p40), and (iii) a nucleic acid encoding VP1 and VP2 from the AAV8 serotype, with the start codon for VP3 capsid protein expression inactivated. This construct can be used to generate haploid AAV8 viruses in vitro. Figure 1B shows an exemplary plasmid map for generating an AAV8-8-rh74 haploid vector. [Figure 1-2] Same as above.

[0086] [Figure 2]Figure 2 shows structural models of AAV8-8-rh10 (left) and AAV8-8-rh74 (right) haploid viruses (four random capsids from one population, as VP subunits can associate differently); red indicates the surface of the virion from the AAV8 VP1 or VP2 capsid protein, green indicates the surface representation from the VP3 capsid protein from the AAVrh10 serotype, and blue indicates the surface representation from the VP3 capsid protein from the AAVrh74 serotype.

[0087] [Figure 3] FIG. 3 shows the simulation and modeling of the tertiary protein structure of the VP3 capsid protein from the AAVrh10 serotype (left) or the AAVrh74 serotype (right).

[0088] [Figure 4] Figure 4: Interface analysis for the parental serotype, AAV88rh.10 and AAV88rh.74 haploids, mutants substituting AAVrh.10 residues for the corresponding residues in AAVrh.74, and the haploids resulting from using these mutants in place of the parental AAVrh.10 VP3. The Y-axis shows the calculated interaction energy between the VP3 subunit and all neighboring subunits with which it is in direct contact. Each complex was minimized before calculating the interaction energy, and this process was repeated 30 times per complex. Mean values ​​of the interaction energies were compared by Wilcoxon pairwise test. Different notation letters above the boxplots indicate statistically significant differences between groups.

[0089] [Figure 5] Figure 5 shows a schematic diagram of a construct for the generation of an AAV8 haploid containing VP1 and VP2 from the AAV8 serotype, showing the modification of two ATG start codons to GTG to result in two amino acid substitutions, M203V and M211V, to prevent translation of the VP3 AAV8 capsid protein. Figure 5 discloses SEQ ID NOS: 28-40, respectively, in order of appearance.

[0090] [Figure 6] Figure 6 shows results from Western blots demonstrating the expression of VP1, VP2, and VP3 proteins from AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 or AAV8-8-VP3KO. The top panel is the schematic diagram shown in Figure 1 for the constructs for the production of AAV8-8-rh10 or AAV8-8-rh74 haploids, and the bottom panel shows Western blot analysis of Pro10 cells infected with AAV8 vector, or AAV8-8-rh10 or AAV8-8-rh74 haploids, and the anti-CAP antibody used to detect VP1 and VP2 from the AAV8 serotype and VP3 from AAVrh74 (lane 1), AAVrh10 (lane 2), or AAV8 (lane 4). Lane 3 shows the absence of VP3 capsid protein expression, indicating that VP3 is not expressed from a construct containing a nucleic acid in which two ATG start codons have been changed to GTG, resulting in the amino acid substitutions M203V and M211V, which prevent translation and expression of the VP3 AAV8 capsid protein.

[0091] [Figure 7] Figure 7 shows the results of a genome protection assay of AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 vector (AAV8-8-8[WT]) or AAV8-VP3KO (AAV8-8-VP3KO).

[0092] [Figure 8] Figure 8 shows the specific productivity of AAV8-8-rh10 or AAV8-8-rh74 haploids as determined by qPCR and ELISA, showing that AAV8-8-rh10 productivity was comparable to AAV8 and AAVrh10 controls. AAV8-8-rh74 had lower productivity levels compared to AAV8-8-rh10 and the control. Mean comparisons are not statistically significant.

[0093] [Figure 9-1]Figures 9A-9B show affinity chromatography and AEX chromatography results for AAV8-8-rh10 or AAV8-8-rh74 haploids. Figure 9A shows affinity chromatography results showing lower SEC 260 / 280 values ​​(see arrows) for AAV8-8-rh74 haploids, suggesting lower packaging efficiency during production. Figure 9B shows AEX chromatography, in which affinity chromatography is followed by an iodixanol density gradient ultracentrifugation (DGUC) process step to separate empty capsids from full capsids, and the iodixanol pool is diluted and loaded onto an AEX column. SEC 260 / 280 values ​​closer to 1.3 (approximately 75% or higher) suggest successful enrichment of full capsid AAV8-8-rh74 (arrow), whereas values ​​of 1.1 indicate lower enrichment. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 9-7] Same as above. [Figure 9-8] Same as above.

[0094] [Figure 10-1]Figures 10A-10D show results for product recovery determined by qPCR and ELISA from AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls. Figure 10A shows product recovery from the AAV8 control, and Figure 10B shows product recovery from the AAVrh10 control. The recovery rates of the current production process for AAV8 and AAVrh10 are 8-12% based on qPCR assays, and the recoveries of the AAV8 and AAVrh10 controls are comparable to each other. Figure 10C shows product recovery from the AAV8-8-rh10 vector containing the luciferase gene, showing that overall recovery rates are comparable to the AAV8 and AAVrh10 controls (Figures 10A and 10B, respectively). Figure 10D shows the product recovery from the AAV8-8-rh74 vector containing the luciferase gene, showing an overall recovery of less than 5%; lower initial productivity leads to lower recovery, but the DSP unit operation, starting with affinity chromatography, reports even lower recovery (approximately 5-12%). [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0095] [Figure 11-1] Figures 11A-11C show electropherogram analyses of AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls. Figure 11A shows the ratios of VP1, VP2, and VP3 for AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls. Figure 11B shows an electropherogram analysis of the AAV8 control (overlay of three individual preparations). Figure 11C shows electropherogram analyses of AAV8-8-rh10 haploids (bottom) and AAV8-8-rh74 haploids (top). [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0096] [Figure 12-1]Figures 12A-12B show representative electropherogram analyses of AAV8-8-rh10 or AAV8-8-rh74 haploids. Figure 12A shows graphs of two representative electropherograms of AAV8-8-rh10 or AAV8-8-rh74 haploids, showing AAV8 reference material and AAV8-8-rh10 or AAV8-8-rh74 virions, respectively. Figure 12B shows graphs of representative electropherograms of AAV5 and AAV9 serotypes, where different serotypes have different relative ratios of the three capsid proteins. [Figure 12-2] Same as above. [Figure 12-3] Same as above.

[0097] [Figure 13-1] Figures 13A-13B show the transduction efficiency results of AAV8-8-rh10 or AAV8-8-rh74 haploids in Pro10 cells compared with AAV8 and AAVrh10 controls. Figure 13A shows a schematic of the protocol for determining the transduction efficacy of each AAV8 haploid vector, in which the plasmids for the generation of the vectors are used to transduce Pro10 production cell lines. Figure 13B shows the transduction efficiency results at 100K MOI for two experiments (left and center graphs) as well as the results of a combined meta-analysis of AAV8-8-rh10 or AAV8-8-rh74 haploids compared with AAV8 and AAVrh10 controls (right graph). These results indicated some differences in the ability of the haploid vectors to transduce Pro10 cells: AAV8-8-rh74 haploids transduced Pro10 cells similarly to the AAV8 control. Statistics were performed by ANOVA + Tukey's test. [Figure 13-2] Same as above.

[0098] [Figure 14-1]Figures 14A-14B show escape from AAV8 neutralizing antibodies by AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls. Figure 14A shows results from two experiments (left and center graphs) and a combined meta-analysis (right graph), suggesting that AAV8-8-rh10 or AAV8-8-rh74 haploids can efficiently escape from neutralizing Ab AAV8 1 / 100 compared to AAV8 and AAVrh10 controls, and therefore show lower % luciferase inhibition compared to controls. Figure 14B shows results from two experiments (left and center graphs) and a combined meta-analysis (right graph) demonstrating the higher efficiency of AAV8-8-rh10 or AAV8-8-rh74 haploids in efficiently evading neutralizing Ab AAV8 1 / 200 compared to AAV8 and AAVrh10 controls, as suggested by lower % luciferase inhibition compared to controls. [Figure 14-2] Same as above.

[0099] [Figure 15] Figure 15 (Experiment 1, Experiment 2, and combined meta-analysis) shows that AAV8-mediated luciferase expression was affected in a dose-dependent manner in the presence of AAV8-neutralizing Abs (sera from AAV8-vaccinated mice), whereas AAV8-8-rh74-mediated luciferase expression was not affected in the presence or absence of AAV8-neutralizing Abs (black bars). Statistics were performed by ANOVA plus Tukey's test.

[0100] [Figure 16] Figure 16 (Experiment 1, Experiment 2, and combined meta-analysis) shows that in the presence of serum from AAV8-vaccinated mice containing AAV8-neutralizing Abs, AAV8-8-rh10-mediated luciferase expression was affected in a similar manner to the AAV8 control (as shown in Figure 15). As suggested by the lower panel of Figure 16, AAV8-neutralizing Abs (AAV8 serum) showed cross-reactivity to AAVrh10. Statistics were performed by ANOVA plus Tukey's test.

[0101] [Figure 17-1] Figures 17A-17B show the results of transduction efficiency of AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls in human fibroblast cell line GM16095 cells. Figure 17A shows a schematic diagram of the protocol for determining the transduction efficacy of each AAV8 haploid vector, and the plasmids for the generation of the vectors used to transduce the neuronal cell line GM16095. Figure 17B shows the results for the transduction efficiency of AAV8-8-rh10 or AAV8-8-rh74 haploids at 100K MOI compared with AAV8 and AAVrh10 controls. These results indicated some differences in the ability of the haploid vectors to transduce GM16095 cells: AAV8-8-Rh74 haploid transduced GM16095 cells significantly more efficiently than AAV8 or AAVrh10 controls, while AAVrh10 was more efficient than AAV8 in this GM16095 cell line. Statistics were performed by ANOVA plus Tukey's test. [Figure 17-2] Same as above.

[0102] [Figure 18-1]Figures 18A-18B show the escape of AAV8 neutralizing antibodies (NAbs) in GM16095 cells by the AAV8-8-rh74 haploid compared to AAV8 and AAVrh10 controls. Figure 18A shows that NAbs in the serum of AAV8-vaccinated mice dose-dependently inhibited AAV8 transduction of GM16095 cells. The AAV8-8-Rh74 haploid vector efficiently escaped anti-AAV8 NAbs, and no difference in luciferase transgene expression was observed in the presence or absence of NAbs. Transduction by the AAV8-8-Rh10 haploid was inhibited by NAbs to the same extent as the AAV8 control. Cross-reactivity of anti-AAV8 NAbs was detected with AAVRH10. Figure 18B shows that NAbs in the serum of mice inoculated with AAV8 at 1 / 100 or 1 / 200 inhibited transduction of GM16095 cells with AAV8 and AAV8-8-rh10, but did not inhibit transduction of GM16095 cells with AAV8-8-rh74 at either 1 / 100 or 1 / 200 concentrations, demonstrating that the AAV8-8-Rh74 haploid vector was able to escape anti-AAV8 NAbs, whereas transduction with the AAV8-8-Rh10 haploid was inhibited by NAbs to the same extent as the AAV8 control. Cross-reactivity of anti-AAV8 NAbs was detected against AAVRh10. [Figure 18-2] Same as above.

[0103] [Figure 19-1]Figures 19A-19B show the efficiency of in vitro transduction of Pro10 cells by AAV8 haploid vectors and the escape of neutralizing antibodies (NABs) in Pro10 cells by AAV8-8-rh74 haploids compared with AAV8 and AAVrh10 controls. Figure 19A shows the higher efficiency of AAV8-8-rh74 transduction of Pro10 cells compared with AAV8 or AAVrh10 at a 100K MOI (left) and in the presence of AAV8 serum at 1 / 100 (center) and 1 / 200 concentrations. Figure 19B shows that NAb in the serum of AAV8-inoculated mice dose-dependently inhibited AAV8 transduction of Pro10 cells. The AAV8-8-Rh74 haploid vector was able to escape anti-AAV8 NAb, and no difference in AAV8-8-rh74-mediated luciferase transgene expression was observed in the presence or absence of NAb. Transduction with the AAV8-8-Rh10 haploid was inhibited by NAb to a similar extent compared to the AAV8 control. Cross-reactivity of anti-AAV8 NAb was detected against AAVRh10. [Figure 19-2] Same as above.

[0104] [Figure 20] Figure 20 is a schematic diagram showing the protocol for analyzing the biodistribution of transduction of AAV8-8-rh10 or AAV8-8-rh74 haploids compared to AAV8 and AAVrh10 controls in mice in vivo. Experiments were performed using four groups of mice, each with five mice; four mice in each group were injected with the experimental vector (experimental mice) and one mouse was left untreated (control mouse). Mice were intravenously injected with 5 x 10 vg / mouse of control AAV8 (control mice), or AAVrh10 or haploids AAV8-8-rh10 or AAV8-8-rh74 (experimental mice).

[0105] [Figure 21-1]Figures 21A-21D show the biodistribution of transduction from AAV8 haploids in vivo as determined by luciferase experiments. Figure 21A shows the ventral biodistribution of luciferase expression after a 30-second exposure at 7 days post-injection (dpi) for AAV8-8-rh10 (Group 2) or AAV8-8-rh74 (Group 3) haploids compared to AAV8 (Group 1) and AAVrh10 (Group 4) controls, demonstrating the distinct biodistribution of the AAV8-8-Rh74 vector, demonstrating both systemic biodistribution and significant distribution of AAV8-8-Rh74 in the brain and spinal cord and across the blood-brain barrier. Figure 21B shows the ventral biodistribution of luciferase expression after a 1-minute light exposure at 7 days post-injection (7 dpi) for AAV8-8-rh10 (Group 2) or AAV8-8-rh74 (Group 3) haploids compared to AAV8 (Group 1) and AAVrh10 (Group 4) controls, demonstrating significantly higher distribution of AAV8-8-rh74 and confirming the results shown in Figure 21A. Figure 21C shows the ventral biodistribution of luciferase expression after an automatic light exposure at 7 days post-injection (7 dpi) for AAV8-8-rh10 (Group 2) or AAV8-8-rh74 (Group 3) haploids compared to AAV8 (Group 1) and AAVrh10 (Group 4) controls, demonstrating basal biodistribution levels in all four groups and thus further supporting the significantly improved biodistribution of AAV8-8-rh74 compared to the other groups. Figure 21D is a graphical representation of the total luminous flux (p / s) of dorsal versus ventral views of luciferase expression after 7 days. Statistics were performed by ANOVA plus Tukey's test (n = 4 mice per group). Similar results were obtained at 14 and 21 days post-injection, with AAV8-8-rh74 consistently demonstrating enhanced systemic biodistribution in vivo compared to other groups, e.g., in the CNS and other tissues. Statistics were performed by ANOVA plus Tukey's test (n = 4 mice per group). Similar results were obtained at 14 and 21 days post-injection, with AAV8-8-rh74 consistently demonstrating enhanced systemic biodistribution in vivo compared to other groups, e.g., in the CNS and other tissues. [Figure 21-2] Same as above. [Figure 21-3] Same as above. [Figure 21-4] Same as above. [Figure 21-5] Same as above. [Figure 21-6] Same as above. [Figure 21-7] Same as above. [Figure 21-8] Same as above. [Figure 21-9] Same as above. [Figure 21-10] Same as above. [Figure 21-11] Same as above. [Figure 21-12] Same as above. [Figure 21-13] Same as above.

[0106] [Figure 22] Figure 22 shows an amino acid sequence alignment of the VP3 capsid protein from AAVrhlO (top sequence) compared to the VP3 capsid protein from AAVrh74 (bottom sequence), showing that the sequences differ in only four regions.

[0107] [Figure 23]Figures 23A-23C show AAVrh.10 (green) relative to AAVrh.74 (cyan). The VP3 domains from both serotypes differ at only five positions: Q417N (red), VV581W (blue), S665N (magenta), and D720E (orange) (using nomenclature / numbering from the amino acid sequence of the VP1 capsid protein). Figure 23A shows the superimposed tertiary structure of the VP3 capsid protein from AAVrh.10 relative to that from AAVrh.74, with residues of interest colored as indicated, while Figure 23B shows the surface of the AAVrh10 capsid and Figure 23C shows the surface of the AAVrh74 capsid, according to the color conventions described above. Figures 23B and 23C clearly show that all positions except position 417 are solvent (and NAb) accessible. Because both Q417 in AAVrh.10 and N417 in AAVrh.74 are buried (no red patch on the surface), they are unlikely to account for any differences in recognition by NAbs. Positions 581 and / or 665 and / or 720 may account for the different recognition of AAVrh.10 compared with AAVrh74 by AcNs, and this is extended to haploids containing them.

[0108] [Figure 24]Figure 24 shows the protease cleavage profiles between AAV8-8-rh10 and AAV8-8-rh74 haploids. The cleavage variables for 19 proteases out of 49 were analyzed. From these, the following groups can be highlighted: (i) cathepsin K (the only lysosomal enzyme in the predicted set). AAVrh.74 has an additional cleavage site. (ii) matrix metallopeptidases-1, -2, -3, and -9. AAVrh.74 loses one site for -1 and -3 and gains one for MMP-2 and -9. As with cathepsin K, it is likely that capsids may come into contact with these enzymes during their normal biodistribution and infection process. (iii) granzymes, elastase, cathepsin G, and caspases: these enzymes may be involved in the immune response to viruses. (iv) pepsin and chymotrypsin are digestive enzymes.

[0109] [Figure 25] Figures 25A-25D show the humoral immune responses of AAV8-8-rh10 or AAV8-8-rh74 haploids compared with AAV8 and AAVrh10 controls. Figure 25A shows anti-AAV8 IgG levels (serum dilution 1 / 1000), and Figure 25B shows anti-AAV8 IgG levels (serum dilution 1 / 5000), demonstrating that significantly reduced anti-AAV8 IgG levels were detected in the serum of mice inoculated with both haploid vectors compared with mice injected with AAV8. No cross-reactivity to AAV8 was observed in the serum from mice inoculated with AAVrh10 at the serum dilutions tested. Figure 25C shows anti-AAVrhlO IgG levels (serum dilution 1 / 1000), and Figure 25D shows anti-AAVrhlO IgG levels (serum dilution 1 / 5000), demonstrating that AAVrhlO was significantly less immunogenic than AAV8 and that no significant differences in anti-AAVrhlO IgG levels were observed between mice inoculated with AAVrhlO and the rest of the experimental groups at the serum dilutions tested. Statistics were performed by ANOVA + Tukey's test (n=4 mice / group).

[0110] [Figure 26-1] Figures 26A-26B show humoral immune responses to AAV8, AAVrhlO, haploid AAV8-8-rh10, and AAV8-8-rh74 in the presence of Nab, e.g., sera from mice inoculated with AAV8 (group 1), mice inoculated with AAV8-8-rh10 (group 2), mice inoculated with AAV8-8-rh74 (group 3), mice inoculated with AAVrhlO (group 4), and untreated controls (group 5), as well as AAV8-mediated GM16095 cell transduction. Figure 26A shows dose-dependent neutralization of AAV8-mediated transduction of GM16095 cells by AAV8 serum, but AAV8-mediated transduction was not neutralized in the presence of serum (1 / 100, 1 / 200, or 1 / 400 dilutions) from mice inoculated with the AAV8-8-rh10 haploid (group 2), or mice inoculated with the AAV8-8-rh74 haploid (group 3), or mice inoculated with AAVrh10 (group 4), or untreated controls (group 5). Figure 26B shows graphs of neutralization by AAV8 serum at a dilution of 1 / 100 (left) or 1 / 200 (right) showing inhibition of luciferase expression from AAV8 alone, but not from AAV8-8-rh10 haploid, AAV8-8-rh74 haploid, or AAVrh10, revealing that AAV8 serum at these concentrations does not neutralize transfection or transduction efficiency by AAV8-8-rh10 or AAV8-8-rh74 haploid. [Figure 26-2] Same as above.

[0111] [Figure 27-1]Figures 27A-27B show humoral immune responses to AAV8, AAVrhlO, haploid AAV8-8-rh10, and AAV8-8-rh74, and AAVrhlO-mediated GM16095 cell transduction in the presence of serum from Nabs, e.g., mice inoculated with AAV8 (group 1), mice inoculated with AAV8-8-rh10 (group 2), mice inoculated with AAV8-8-rh74 (group 3), mice inoculated with AAVrhlO (group 4), and untreated controls (group 5). Figure 27A shows that neutralization of GM16095 cell transduction with AAVrhlO was not detected in serum from mice inoculated with AAVrhlO at any dilution tested (1 / 100, 1 / 200, and 1 / 400) (group 4). Figure 27B shows graphs of neutralization by AAVrh10 at a dilution of 1 / 100 (left) or 1 / 200 (right) showing inhibition of luciferase expression from AAV8, AAV8-8-rh10, and AAV8-8-rh74, suggesting their cross-reactivity to AAVrh10 serum; Figure 27B shows graphs of neutralization by AAVrh10 serum at a dilution of 1 / 100 (left) or 1 / 200 (right) showing inhibition of luciferase expression from AAV8, AAV8-8-rh10, and AAV8-8-rh74, suggesting their cross-reactivity to AAVrh10 serum. [Figure 27-2] Same as above.

[0112] [Figure 28-1]Figure 28 shows a schematic diagram of the construct for generating the AAV8-8-rh74 haploid (top construct) or the AAV8-8-rh10 haploid (bottom construct). An amino acid sequence alignment of the VP3 capsid protein from AAVrh10 (top sequence) compared to the VP3 capsid protein from AAVrh74 (bottom sequence) is also shown, demonstrating that these sequences differ only in four regions: Q214N, VV378W, S462N, and D517E, using the nomenclature / numbering from the amino acid sequence of the VP3 capsid protein from AAVrh10 (also shown in Figure 22). The corresponding amino acid positions according to the VP1 capsid protein of AAVrh10 are Q417N, V581(del), V582W, S665N, and D720E. [Figure 28-2] Same as above.

[0113] [Figure 29-1]Figures 29A-29C show the production yields of AAV8-8-rh10 or AAV8-8-rh74 haploids containing amino acid modifications. Figure 29A shows a greater than four-fold increase in yield of AAV8-8-rh74(W581VV) (also referred to as AAV8-8-rh74vv) compared to the yield from AAV8-8-rh74 (unmodified), which is similar to the yield of AAV8-8-rh10. In Figure 29A, AAVrh74vv is represented as AAV8-8-rh74 581-W582V (used interchangeably with W581VV throughout this application). Modifications of the rh74 VP3 protein, N417Q, N664S, and E719D, did not improve yield (see AAV8-8-rh74(N417Q), AAV8-8-rh74(N664S), and AAV8-8-rh74(E719D)). Results from three independent transduction experiments are shown. Figure 29B shows that AAV8-8-rh10(V581del) and AAV8-8-rh10(V582W) significantly reduced yield compared to unmodified AAV8-8-rh10, while AAV8-8-rh10(S665N) and AAV8-8-rh10(D720E) did not significantly affect yield compared to unmodified AAV8-8-rh10. Taken together, Figures 29A and 29B confirm, through forward and reverse mutations, that amino acids 581 and 582 of the VP3 capsid protein from Rh10 are important for virus production. Figure 29C further confirms, using ITR-qPCR analysis after DNase and proteinase K treatment, that Q417N, S665N, and D720E had little effect on virus production, while V581del and V582W reduced virus production compared to unmodified AAV8-8-rh10; using ITR-qPCR analysis after DNase and proteinase K treatment, further confirms that Q417N, S665N, and D720E had little effect on virus production, while V581del and V582W reduced virus production compared to unmodified AAV8-8-rh10. [Figure 29-2] Same as above.

[0114] [Figure 30-1] Figures 30A-30C show gene expression of the luciferase transgene in brain and spinal cord tissues from mice intravenously administered the AAV8-8-rh74 vector, confirming that the AAV8-8-rh74 vector crosses the blood-brain barrier (BBB). Figure 30A shows the protocol for assessing the biodistribution of haploid AAV8-8-Rh74 or AAV8-8-Rh10 vectors in the brain, spinal cord, and small intestine, demonstrating that tissues were collected 28 days after i.v. administration of 2.5 x 10 vg / kg of AAV vector and luciferase transcripts were assessed by RT-PCR. Figure 30B shows the results of RT-PCR of luciferase in brain tissue from mice 28 days after i.v. administration of 2.5 x 10 vg / kg of either AAV8-8-Rh10, AAV8, AAV8-8-Rh74, or AAV8-8-Rh10 (n=4). Figure 30C shows the results of RT-PCR for luciferase in spinal cord tissue from mice 28 days after iv administration of 2.5 x 1012 vg / kg of either AAVRh10, AAV8, AAV8-8-Rh74, or AAV8-8-Rh10 (n=4). [Figure 30-2] Same as above. [Figure 30-3] Same as above.

[0115] [Figure 31] Figure 31 shows the results of RT-PCR to determine gene expression of a luciferase transgene in the small intestine of mice 28 days after intravenous administration of AAVRh10, AAV8, AAV8-8-Rh74, or AAV8-8-Rh10 (n=4), demonstrating that the AAV8-8-rh74 vector has significant tropism compared to AAVRh10, AAV8, and AAV8-8-Rh10 and efficiently transduces the small intestine.

[0116] [Figure 32-1] FIG. 32 shows Tables 9, 10, 11 and 12. [Figure 32-2] Same as above. [Figure 32-3] Same as above. [Figure 32-4] Same as above. [Figure 32-5] Same as above. [Figure 32-6] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0117] Detailed Description of the Invention The present invention will now be described with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. However, the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0118] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The terminology used in describing the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, accession numbers and other references mentioned herein are incorporated herein by reference in their entirety.

[0119] All amino acid position designations in the AAV capsid viral structural proteins in the present description and appended claims are with respect to VP1 capsid subunit numbering (native AAV8 VP1 capsid protein: GenBank accession number AF513852.1, protein ID: AAN03856.1). Those skilled in the art will understand that the modifications described herein, when inserted into the AAV cap gene, can lead to modifications of the structural viral proteins VP1, VP2, and / or VP3 that make up the capsid subunit. Alternatively, the capsid subunits can be expressed independently to achieve modifications of only one or two of the capsid subunits (VP1, VP2, VP1+VP2).

[0120] In particular, in previous studies, the inventors demonstrated that polyploid (e.g., haploid) AAV vectors using VPs from multiple serotypes result in different biodistributions and higher transduction in specific tissues, such as the liver.

[0121] The technology herein generally relates to homogeneous populations of rational polyploid adeno-associated virus (AAV) particles, virions, and viral capsids comprising VP3 structural proteins from any AAV serotype that cross the blood-brain barrier (BBB), wherein the polyploid AAV virions cross the BBB upon systemic or intrathecal administration and / or transduce cells of the BBB or cerebrovascular (BBV) endothelial cells (BBV-ECs) and / or blood components that cross the BBB. In some embodiments, the VP3 capsid protein is from a non-human primate, and in some embodiments, the VP3 capsid protein is an AAV rhesus serotype. In some embodiments, the rational polyploid AAV virions comprise, in addition to the VP3 protein, at least one of VP1 and / or VP2 viral structural proteins. In certain embodiments, rational polyploid AAV virions comprise a VP1 capsid protein from AAV8 and at least one VP3 capsid protein from any AAV serotype that crosses the BBB.

[0122] In some embodiments, the homogenous population of rational polyploid adeno-associated virus (AAV) particles disclosed herein that are capable of crossing the BBB and / or transducing cells of the BBB or cerebrovascular (BBV) endothelial cells (BBV-ECs) and / or blood components that cross the BBB may be selected from any of AAV8-8-rh10, AAV8-8-rh74, AAV8-8-rh74vv, AAV8-8-rh10LP2, AAV8-8-rh74LP2, AAV8-8-rh74vvLP2. In some embodiments, such AAV haploid virions that cross the BBB and / or transduce cells of the BBB or BBV-ECs or cells that cross the BBB may comprise AAV8 VP1 and VP2 structural proteins (e.g., SEQ ID NOs: 7 and 8), or may comprise the modified protein of SEQ ID NO: 7 or 8 and a VP3 protein selected from any of rh10 VP3 (SEQ ID NO: 1), rh74 VP3 (SEQ ID NO: 3), rh74vv VP3 (SEQ ID NO: 2), rh10-LP2 VP3 protein (SEQ ID NO: 14), rh74-LP2 VP3 protein (SEQ ID NO: 17), and rh74vv-LP2 VP3 protein (SEQ ID NO: 15), or a VP3 protein having an amino acid sequence that is at least 85% sequence identical to any of SEQ ID NOs: 1, 2, 3, 14, 15, and 18.

[0123] The technology described herein is based, in part, on the discovery that the use of the VP3 structural protein, also referred to herein as "capsid protein," from any AAV serotype that efficiently crosses the BBB alters the biodistribution and transduction efficiency of the vector after systemic or intrathecal administration, and in particular, in some embodiments, exhibits an increased ability of the AAV vector to cross the BBB and transduce one or more tissues in the CNS or peripheral nervous system (PNS). In some embodiments, it exhibits increased transduction of BBB endothelial cells (BBB ECs) and / or components of the BBB, or increased transduction of endothelial cells of the cerebrovascular (BBV), or increased transduction of blood components that cross the BBB. In some embodiments, the rational polyploid vector comprises a VP3 viral structural protein from any serotype selected from the group consisting of AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43. In some embodiments, rational polyploid vectors comprise the VP3 viral structural protein from any non-primate AAV serotype, such as a rhesus AAV serotype.

[0124] For illustrative purposes only, the Examples demonstrate exemplary polyploid, e.g., haploid, vectors with increased ability to cross the BBB upon systemic or intrathecal administration. For illustrative purposes only, such haploid vectors include VP3 from any AAV serotype that efficiently crosses the BBB and VP1 and / or VP2 from the AAV8 serotype. The AAV VP1 and / or VP2 structural proteins can be VP1 or VP2 from any AAV serotype selected from Table 1. Thus, exemplary haploid vectors are described herein, including capsid protein VP1 from the AAV8 serotype and at least one capsid protein VP3 from any AAV serotype that crosses the BBB and / or from a non-human primate, but not the AAV8 serotype.

[0125] Preferably, such populations of rational polyploid virions are substantially homogeneous. In some embodiments, the rational polyploid virions of the present invention may comprise a VP2 capsid protein, wherein the VP2 capsid protein is from any serotype or is a chimeric VP2 protein thereof, or the VP2 capsid protein is from any serotype that is the same as the serotype from which VP3 is derived, or alternatively, the VP2 capsid protein is from a serotype different from the serotype from which VP3 is derived.

[0126] In some embodiments, rational polyploid virions as disclosed herein are produced that contain VP1 from the AAV8 serotype and at least one VP3 capsid protein, where the VP3 is not from AAV8 but is selected from any serotype that crosses the BBB and / or is a non-human primate AAV serotype. I. Definition

[0127] The following terms are used in the description and appended claims:

[0128] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0129] Furthermore, the term "about," when used herein in reference to a measurable value, e.g., amount of polynucleotide or polypeptide sequence length, dose, time, temperature, etc., is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0130] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0131] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim should be construed to include the specified materials or steps recited in the claim, as well as those "which do not materially affect the basic and novel characteristics" of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in original). See also MPEP § 2111.03. Thus, the term "consisting essentially of," when used in the claims of the present invention, is not intended to be construed as equivalent to "comprise." Unless the context dictates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination.

[0132] Furthermore, the present invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted.

[0133] To further illustrate, for example, if the specification indicates that a particular amino acid may be selected from A, G, I, L, and / or V, this language also indicates that the amino acid may be selected from any subset of these amino acids, e.g., A, G, I, or L; A, G, I, or V; A or G; L only, etc., as if each such subcombination were explicitly set forth herein. Furthermore, such language also indicates that one or more of the specified amino acids may be waived (e.g., by a negative disclaimer). For example, in certain embodiments, the amino acid is not A, G, or I; is not A; is not G or V, etc., as if each such possible waiver were explicitly set forth herein.

[0134] As used herein, the terms "reduce," "reduces," "reduction," and similar terms refer to a decrease of at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, or more. As used herein, the terms "enhance," "enhances," "enhancement," and similar terms refer to an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more. The enhanced transduction ability of the rational polyploid virions of the present invention across the blood-brain barrier is compared to AAV virions lacking the ability to cross the blood-brain barrier. The enhanced transduction ability of the rational polyploid virion across the blood-brain barrier is at least 25%, 50%, 60%, 70%, 80%, 90%, or 95%, or 100% greater, or at least 1.2-fold, or at least 1.5-fold, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or greater, compared to that of an AAV virion lacking the ability to cross the blood-brain barrier. Non-limiting examples of AAV serotypes lacking the ability to cross the blood-brain barrier include AAV2, AAV5, and AAV8. In one embodiment, the population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​compared to AAV serotypes that do not efficiently cross the blood-brain barrier, such as AAV8, AAV2, or AAV5. In one embodiment, the population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​compared to AAV8. In one embodiment, the population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​compared to AAV2. In another embodiment, the population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​compared to AAV5. In some embodiments, the population of rational polyploid AAV virions has improved biodistribution in the brain and spinal cord compared to AAV8. The term "parvovirus," as used herein, encompasses the Parvoviridae family, including autonomously replicating parvoviruses and dependoviruses.Autonomous parvoviruses include members of the Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus genera. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus, and any other autonomous parvoviruses now known or later discovered. Other autonomous parvoviruses are known to those skilled in the art. See, for example, Bernard N. Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

[0135] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Several relatively new AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) Virology 78:6381-6388; Morris et al., (2004) Virology 33-375-383; and Table 3).

[0136] The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC See, e.g., 001540, AF513851, AF513852, AY530579, the disclosures of which are incorporated herein by reference for their teaching of parvovirus and AAV nucleic acid and amino acid sequences.For example, Srivistava et al., (1983) J. Virology 45:555;Chiarini et al., (1998) J. Virology 71:6823;Chiarini et al., (1999) J. Virology 73:1309;Bantel-Schaal et al., (1999) J. Virology 73:939;Xiao et al., (1999) J. Virology 73:3994;Muramatsu et al., (1996) Virology 221:208;Shade et al., (1986) J. Virol. 58:921;Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854;Moris et al., (2004) Virology 33-:375-383; 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 for their teaching of parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1.

[0137] The capsid structure of autonomous parvoviruses and AAV is described in more detail in Bernard N. Fields et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also the crystal structure descriptions of AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99:10405-10), AAV4 (Padron et al., (2005) J. Virol. 79:5047-58), AAV5 (Walters et al., (2004) J. Virol. 78:3361-71), and CPV (Xie et al., (1996) J. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251:1456-64).

[0138] The term "tropism," as used herein, refers to the preferential entry of a virus into certain cells or tissues, optionally followed by expression (e.g., transcription, and optionally translation) of sequences carried by the viral genome in the cell, e.g., in the case of a recombinant virus, expression of a heterologous nucleic acid of interest.

[0139] As used herein, "systemic tropism" and "systemic transduction" (and synonyms) refer to a viral capsid or viral vector of the invention that exhibits tropism for and / or transduces tissues throughout the body (e.g., brain, lung, skeletal muscle, heart, liver, kidney, and / or pancreas). In embodiments of the invention, systemic transduction of the central nervous system (e.g., brain, neurons, etc.) is observed.

[0140] As used herein, "selective tropism" or "specific tropism" refers to the delivery and / or specific transduction of a viral vector to certain target cells and / or certain tissues.

[0141] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms may be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500%, or more of the transduction or tropism, respectively, of the control). In some embodiments, the control that serves as a reference for determining whether an AAV virion can "efficiently cross the blood-brain barrier" is an AAV virion that lacks the ability to cross the blood-brain barrier. In certain embodiments, the rational polyploid viral vectors described herein efficiently transduce or have efficient tropism for the CNS or peripheral nervous system (PNS), including neuronal and non-neuronal cells. Suitable controls will depend on various factors, including the desired tropism and / or transduction profile. Those skilled in the art can determine whether the rational polyploid AAV vectors disclosed herein can efficiently cross the blood-brain barrier better than AAV2 under the same conditions by monitoring transduction across CNS regions, such as the brain, as described, for example, in Molecular Therapy, Vol. 19, No. 8, Aug. 2011, the entire contents of which are incorporated herein by reference. Monitoring can be performed by any method known to those skilled in the art, including RT-PCR analysis of brain and spinal cord tissues, Western blot analysis of brain and spinal cord tissues, or immunohistochemical examination of brain and spinal cord tissues in animal models, as well as in vivo analysis of bioluminescence from luciferase-expressing rational polyploids in animal models. Serotypes that efficiently cross the blood-brain barrier are preferably better than AAV6 at crossing the blood-brain barrier and transducing CNS regions, such as the brain. In one embodiment, the population is able to cross the blood-brain barrier and transduce regions of the CNS better than AAV5.In some embodiments, the population exhibits enhanced transduction into one or more of the subject's cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord compared to AAV8, a non-haploid AAV particle that lacks the ability to efficiently cross the blood-brain barrier. In some embodiments, the rational polyploid population is administered to a subject by intravenous injection, intrathecal injection, or intravascular injection into the brain. The enhanced transduction ability of the rational polyploid virions disclosed herein to transduce CNS or brain regions is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% greater, or at least 1.2-fold, or at least 1.5-fold, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold greater, or greater, compared to that of AAV2 or AAV5 virions under the same conditions.

[0142] Similarly, whether a virus "does not efficiently transduce" a target tissue, or whether it "does not have efficient tropism" for a target tissue, or similar expressions, can be determined by reference to a suitable control. In certain embodiments, a viral vector does not efficiently transduce (i.e., does not have efficient tropism for) liver, kidney, gonad, and / or germ cells. In certain embodiments, transduction (e.g., undesired transduction) in a tissue (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the transduction level in a desired target tissue (e.g., skeletal muscle, diaphragm muscle, cardiac muscle, and / or cells of the central nervous system).

[0143] In some embodiments of the invention, AAV particles comprising capsids of the invention can demonstrate multiple phenotypes: efficient transduction of certain tissues / cells, and very low levels of transduction (e.g., reduced transduction) of certain tissues / cells, which is undesirable.

[0144] As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise indicated.

[0145] A "polynucleotide" is a sequence of nucleotide bases, which may be RNA, DNA, or a DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is either a single-stranded or double-stranded DNA sequence.

[0146] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus with which it is typically found associated, e.g., cellular or viral structural components or other polypeptides or nucleic acids. In representative embodiments, the "isolated" nucleotide is at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more enriched relative to the starting material.

[0147] Similarly, an "isolated" polypeptide refers to a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus with which it is typically found associated, e.g., cellular or viral structural components or other polypeptides or nucleic acids. In representative embodiments, the "isolated" polypeptide is at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more enriched compared to the starting material.

[0148] "Isolated cells" refers to cells that are separated from other components that they normally associate with in their natural state. For example, isolated cells can be cells in culture medium and / or cells in the pharmaceutically acceptable carrier of the present invention. Thus, isolated cells can be delivered and / or introduced into a subject. In some embodiments, isolated cells can be cells that are removed from a subject, manipulated ex vivo as described herein, and then returned to the subject.

[0149] The population of virions can be generated by any of the methods described herein. In one embodiment, the population comprises at least 10 1 In one embodiment, the population is at least 10 virions. 2 virions, at least 10 3 virions, at least 10 4 virions, at least 10 5 virions, at least 10 6 virions, at least 10 7 virions, at least 10 8 virions, at least 10 9 virions, at least 10 10 virions, at least 10 11 virions, at least 10 12 virions, at least 10 13 virions, at least 10 14 virions, at least 10 15 virions, at least 10 16 virions, or at least 10 17 A population of virions can be heterogeneous or homogeneous (e.g., substantially homogeneous or completely homogeneous).

[0150] A "substantially homogeneous population," as that term is used herein, refers to a population of nearly identical virions with few to no contaminating virions (non-identical) within the population. A substantially homogeneous population is at least 90% identical virions (e.g., desired virions), and can also be at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical virions.

[0151] A population of virions that is completely homogeneous contains only identical virions.

[0152] As used herein, "isolating" or "purifying" (or grammatical equivalents) a viral vector or viral particle or population of viral particles means that the viral vector or viral particle or population of viral particles has been at least partially separated from at least some of the other components in the starting material. In representative embodiments, the "isolated" or "purified" viral vector or viral particle or population of viral particles is at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more enriched compared to the starting material.

[0153] A "therapeutic polypeptide" is a polypeptide that can alleviate, reduce, prevent, delay, and / or stabilize symptoms that occur as a result of the absence or deficiency of a protein in a cell or subject, and / or otherwise provide a benefit to the subject, e.g., an anti-cancer effect, or improved survival of a transplant, or induction of an immune response.

[0154] The terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean to lessen the severity of, at least partially improve or stabilize, the subject's condition, and / or to achieve some alleviation, reduction, decrease or stabilization of at least one clinical symptom, and / or to slow the progression of the disease or disorder.

[0155] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of, and / or reducing the severity of, a disease, disorder, and / or clinical symptom in a subject compared to what would occur in the absence of the methods of the present invention. Prevention can be complete prevention, e.g., the complete absence of a disease, disorder, and / or clinical symptom. Prevention can also be partial prevention, such that the appearance and / or severity of the onset of a disease, disorder, and / or clinical symptom in a subject is substantially less than what would occur in the absence of the present invention.

[0156] " Therapeutic effective amount " as used herein is the amount that is sufficient to bring about some improvement or benefit to the subject.In other words, " therapeutic effective amount " is the amount that will bring about some relief, alleviation, reduction or stabilization of at least one clinical symptom in the subject.It will be understood by those skilled in the art that the therapeutic effect does not need to be complete or curative, as long as some benefit is provided to the subject.

[0157] A "prophylactically effective" amount, as used herein, is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, and / or to reduce the severity and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, compared to that which would occur in the absence of the method of the present invention. It will be understood by those skilled in the art that the level of prevention need not be complete, provided that some prophylactic benefit is provided to the subject.

[0158] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein and refer to a nucleic acid sequence that does not naturally occur in a virus. Generally, a heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame encoding a polypeptide and / or untranslated RNA of interest (e.g., for delivery to a cell and / or subject).

[0159] As used herein, the terms "viral vector," "vector," or "gene delivery vector" refer to a viral (e.g., AAV) particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within the virion. Alternatively, in some contexts, the term "vector" may be used to refer to the vector genome / vDNA alone.

[0160] A "rAAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) containing one or more heterologous nucleic acid sequences. rAAV vectors generally require only the terminal repeats (TRs) in cis to produce virus. All other viral sequences are not essential and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, rAAV vector genomes will retain only one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and nonstructural protein coding sequences may also be provided in trans (e.g., from a vector such as a plasmid or by stably integrating sequences into the packaging cell). In embodiments of the invention, the rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), and optionally two TRs (e.g., two AAV TRs), which are typically at the 5' and 3' ends of the vector genome and will be adjacent to, but not necessarily contiguous with, the heterologous nucleic acid. The TRs may be the same as or different from one another.

[0161] The term "terminal repeat" or "TR" includes any viral or synthetic terminal repeat sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function, such as replication, viral packaging, integration, and / or proviral rescue). A TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR sequence, such as that of another parvovirus (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., an SV40 hairpin that serves as an SV40 origin of replication), can be used as a TR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Furthermore, a TR can be partially or completely synthetic, such as a "double D sequence" as described in U.S. Patent No. 5,478,745 to Samulski et al.

[0162] An "AAV terminal repeat" or "AAV TR" can be from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any other AAV currently known or later discovered (see, e.g., Table 1). An AAV terminal repeat need not have its native terminal repeat sequence (e.g., the native AAV TR sequence may be altered by insertion, deletion, truncation, and / or missense mutation), so long as the terminal repeat mediates a desired function, such as replication, viral packaging, integration, and / or proviral rescue. The AAV proteins VP1, VP2, and VP3 are capsid proteins that interact with each other to form the icosahedral-symmetric AAV capsid. VP1.5 is an AAV capsid protein described in U.S. Patent Application Publication No. 2014 / 0037585. In some embodiments of the present invention, the AAV ITR is 145 bp. In some embodiments, the AAV ITRs are smaller than 145 bp, hi some embodiments, the AAV ITRs are 130 bp.

[0163] The viral vectors of the present invention may further be "targeted" viral vectors (e.g., with directed tropism) and / or "hybrid" parvoviruses (i.e., in which the viral TR and viral capsid are from different parvoviruses) as described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0164] The viral vectors of the present invention may further be double-stranded parvovirus particles as described in International Patent Publication No. WO 01 / 92551, the disclosure of which is incorporated herein by reference in its entirety. Thus, in some embodiments, a double-stranded (duplex) genome may be packaged into the viral capsids of the present invention.

[0165] In addition, the viral capsid or genomic elements may contain other modifications, including insertions, deletions and / or substitutions.

[0166] As used herein, a "chimeric" viral structural protein refers to an AAV viral structural protein (capsid) that has been modified by the substitution of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues within the amino acid sequence of the capsid protein relative to the wild-type, as well as the insertion and / or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues within the amino acid sequence relative to the wild-type. In some embodiments, complete or partial domains, functional regions, epitopes, etc. from one AAV serotype may be substituted for corresponding wild-type domains, functional regions, epitopes, etc. from a different AAV serotype, in any combination, to produce a chimeric capsid protein of the invention. In other embodiments, the substitutions are all from the same serotype. In other embodiments, the substitutions are all from AAV or synthetic. Production of chimeric capsid proteins can be performed according to protocols well known in the art, and numerous chimeric capsid proteins have been described herein as well as in the literature, which can be included in the capsids of the present invention. In some embodiments, the rational polyploid comprises at least one chimeric viral structural protein. In this aspect, the viral structural protein is generated with the N-terminus of one AAV serotype and the C-terminus of another AAV serotype. In some embodiments, the rational polyploid does not comprise a chimeric viral structural protein.

[0167] The "modified" viral structural protein may be a non-capsid protein or a modified or chemically modified viral structural protein, such as the addition of a non-naturally occurring or synthetic amino acid, or the substitution of an amino acid with a non-naturally occurring amino acid, as well as a structural capsid protein that is a chemical modification of one or more existing amino acids of an AAV capsid protein. Surface modifications to capsid proteins are disclosed in U.S. Patent Nos. 10,294,281, 9,409,953, and U.S. Patent Application Publication No. 2018 / 0105559, which are incorporated herein by reference in their entirety. In some embodiments, the surface modification may include a targeting protein that targets the CNS, such as those disclosed in WO2020028751, particularly Table 2 of WO2020028751, which are incorporated herein by reference in their entirety. In certain embodiments, rational engineering and / or mutation methods are used to modify and / or target peptides that exhibit enhanced transduction of target tissues (e.g., CNS or PNS). Targeting peptides for use and modification of one or more capsid proteins of the rational polyploid vectors disclosed herein can be identified and / or designed by any method known in the art, for example, using the CREATE system as described in Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)) and International Patent Application Publication Nos. WO2015038958 and WO2017100671, which are incorporated herein in their entireties.

[0168] In an alternative embodiment, virion particles can be assembled in which at least one viral protein selected from the group consisting of AAV capsid proteins VP1, VP2, and VP3 is different from at least one of the other viral proteins required to form virion particles capable of encapsidating the AAV genome. For each viral protein present (VP1, VP2, and / or VP3), the protein is of the same type (e.g., all AAV2 VP1). In one example, at least one of the viral proteins is a chimeric viral protein, and at least one of the other two viral proteins is not chimeric. In one embodiment, VP1 and VP2 are chimeric, and only VP3 is non-chimeric. For example, only viral particles composed of VP1 / VP2 from chimeric AAV2 / 8 (N-terminus of AAV2 and C-terminus of AAV8) paired with only VP3 from AAV2; or only chimeric VP1 / VP2 28m-2P3 (N-terminus from AAV8 and C-terminus from AAV2 without mutation of VP3 start codon) paired with only VP3 from AAV2. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from a completely different serotype. For example, only chimeric VP1 / VP2 28m-2P3 paired with VP3 from AAV3. In another example, there is no chimeric one.

[0169] As used herein, the term "amino acid" includes any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Naturally occurring levorotatory (L-) amino acids are shown in Table 10.

[0170] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 12) and / or an amino acid that has been modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0171] Additionally, non-naturally occurring amino acids can be "unnatural" amino acids as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.

[0172] As used herein, the term "homologous recombination" refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Homologous recombination also generates new combinations of DNA sequences. These new combinations of DNA represent genetic diversity. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of viruses.

[0173] Viral infection of a host can stimulate the host's immune defense system to protect the infected host from the virus. One of the immune responses activated by the host to protect itself from foreign attack is the humoral immune response, which generates antibody-mediated immunity.

[0174] As used herein, the term "humoral immunity" refers to the antibody-mediated beta cell immune system (as opposed to cellular immunity), which is mediated by macromolecules found in extracellular fluids, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. In particular, it refers to the antibody-mediated immune response of a host.

[0175] As used herein, "antigenic" or "antigenicity" is used interchangeably with "immunogenic" and refers to the ability of a substance, e.g., a rational polyploid virion, to induce a specific immune response. It also refers to the extent to which a substance is capable of stimulating an immune response, e.g., the ability of a substance to bind to an antibody or to a T cell receptor or to a B cell.

[0176] As used herein, the term "neutralizing antibody" is used interchangeably with "Nab" and refers to an antibody that specifically binds to an epitope critical to viral function and interferes with viral infectivity, e.g., blocks AAV virion entry into host cells. Within this definition, a neutralizing antibody (NAb) refers to an antibody that protects cells from antigens or infectious agents by inhibiting or neutralizing any biological effects of those antigens or infectious agents. Generally, an antibody binds to an antigen and transmits a signal to white blood cells that the antigen has targeted (flagged). Flagged antigens are processed and subsequently destroyed, whereas neutralizing antibodies neutralize the biological effects of the antigen itself. NAbs can be broadly neutralizing antibodies (bNAbs) that act against multiple viral serotypes, or specific NAbs that specifically recognize one serotype.

[0177] "Neutralization" of viruses, particularly AAV capsids and AAV serotypes, is defined herein as the abrogation of viral infectivity in vitro or in vivo by binding of a neutralizing compound (e.g., an antibody) to a viral serotype and / or preventing cell surface binding and interaction with AAV. For the purposes of the present invention, this definition does not include blocking infection by neutralizing antibodies that bind to viral receptors on the (host) cell surface.

[0178] In some examples, neutralizing potency is determined by measuring the activity of a reporter gene product (e.g., luciferase, GFP). The neutralizing potency of an antibody against a specific AAV serotype can be at least 50%, for example, at least 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

[0179] As used herein, the term "blood-brain barrier" or "BBB" refers to a highly selective, semi-permeable boundary of endothelial cells that prevents solutes in circulating blood from non-selectively crossing and entering the extracellular fluid of the central nervous system where neurons reside. The blood-brain barrier is formed by endothelial cells in the capillary walls, astroglial foot processes that ensheath the capillaries, and pericytes embedded in the capillary basement membrane. This system allows the passage of some molecules by passive diffusion and also allows the selective active transport of various nutrients, ions, organic anions, and macromolecules, such as glucose, water, and amino acids, that are crucial for neuronal function.

[0180] As used herein, "cerebrovascular" or "BBV" refers to the blood vessels and capillaries in the brain that are part of the cerebral circulation and have BBB function.

[0181] As used herein, the phrase "cells of the BBB" refers to any cell that is part of or a component of the BBB, including endothelial cells of the capillary walls of the BBB, astrocytic foot processes that ensheath the capillaries, and pericytes embedded in the capillary basement membrane.

[0182] As used herein, the phrase "endothelial cells of the BBB" is used interchangeably herein with "BBB ECs" or "BBB endothelial cells" and refers to the endothelial cells of the capillary walls of the BBB.

[0183] As used herein, the phrase "cells that cross the BBB" refers to any cell that can cross an intact BBB, i.e., a BBB that is not leaky or compromised. Cells that can cross the BBB include, but are not limited to, perivascular pericytes, macrophages, T cells, and monocytes.

[0184] As used herein, "blood components" as used herein refers to cells in the blood, including platelets (thrombocytes), red blood cells (RBCs) (erythrocytes), and white blood cells, which include lymphocytes, monocytes, eosinophils, basophils, and neutrophils.

[0185] As used herein, the terms "gene editing," "genome editing," or "genome engineering" refer to a type of genetic engineering in which DNA is inserted, deleted, or replaced in the genome of an organism using engineered nucleases, or "molecular scissors." These nucleases create site-specific double-strand breaks (DSBs) at desired locations within the genome.

[0186] As used herein, the term "gene delivery" refers to the process by which foreign DNA is transferred into host cells for gene therapy applications.

[0187] As used herein, "CRISPR" stands for Clustered Regularly Interspaced Short Palindromic Repeats, which is a hallmark of the bacterial defense system that underlies CRISPR-Cas9 genome editing technology.

[0188] As used herein, the term "zinc finger" refers to a small protein structural motif characterized by the coordination of one or more zinc ions to stabilize the folded structure. II. Rational Haploid or Polyploid AAV Vectors in General

[0189] As used herein, a rational polyploid, e.g., a rational haploid, refers to a virion formed from viral structural proteins VP1, VP2, or VP3 derived from at least two different AAV serotypes, where each of VP1, VP2, or VP3 is derived from only one parent AAV serotype. A non-rational haploid may refer to a chimeric or mosaic haploid. In one embodiment, the viral structural proteins of the parent serotype may be modified viral structural proteins or chimeric. In one embodiment, the parent serotype is not a chimeric serotype. In some embodiments, the modified parent AAV serotype contains one or more amino acid insertions, deletions, or substitutions. A rational polyploid virion, e.g., a rational haploid, of the present invention is not a mosaic virion.

[0190] In some embodiments, the AAV hybrid particles disclosed herein can be synthetic AAV hybrid viral vectors designed to display a variety of desirable phenotypes suitable for different in vitro and in vivo applications. Thus, in one embodiment, the present invention provides AAV hybrid particles or virions. In one embodiment, the present invention provides a substantially pure population of AAV hybrid particles or virions.

[0191] The present invention provides a number of synthetic viral vectors that display a variety of desirable phenotypes suitable for different in vitro, in vivo and clinical applications.

[0192] In particular, the present invention is based on the unexpected discovery that combining at least VP3 capsid protein exclusively from any AAV serotype that efficiently crosses the BBB with VP1 and / or VP2 capsid proteins exclusively from a different serotype (e.g., VP1 and / or VP2 capsid proteins from an AAV serotype selected from Table 1) can result in improved AAV virions with multiple desirable phenotypes in each individual capsid, including, but not limited to, increased systemic delivery and increased tropism, and distinct antigenic profiles, such as the ability to escape neutralizing antibodies (Nabs) in vivo following administration.

[0193] In some embodiments, the rational polyploid vectors disclosed herein have biodistribution in the CNS and / or PNS due to the rational polyploid's ability to cross the BBB. In some embodiments, the rational polyploid vectors disclosed herein have biodistribution in the CNS and / or PNS due to their ability to transduce brain vasculature (BBV) and / or blood components. In some embodiments, the rational polyploid vectors disclosed herein have biodistribution in the CNS and / or PNS due to transduction of brain vasculature (BBV) and / or blood components, e.g., cells in the blood, which allows delivery of AAV-transduced cells to the brain via the cerebral circulation. In some embodiments, the rational polyploid vectors disclosed herein can transduce cells that cross endothelial cells, including perivascular pericytes and macrophages, as well as other immune cells, e.g., T cells and blood monocytes. That is, in some embodiments, rational polyploid AAV vectors have biodistribution in the brain by an indirect route—the AAV polyploid vectors disclosed herein can reach the brain indirectly by transducing cells that enter the brain and / or by transducing brain vasculature (BBV), which contains endothelial cells within the brain. In some embodiments, rational polyploid vectors disclosed herein can be taken up by neuronal and / or non-neuronal cells in contact with brain vasculature or other organs, for example, by retrograde transport from peripheral organs to the CNS via autonomous or peripheral nerve fibers. In some embodiments, rational polyploids of the present invention exhibit enhanced binding to brain microvascular endothelial cells (BMVECs) compared to those of AAV8. In some embodiments, rational polyploids of the present invention exhibit enhanced binding to brain microvascular endothelial cells (BMVECs) compared to those of AAV2. Binding to BMVECs is described in Molecular Therapy, Methods & Clinical Development, Vol. 20, March 2021, the entire contents of which are incorporated herein by reference.In some embodiments, the rational polyploids of the present invention can diffuse, transcytose, or shuttle across an endothelial barrier, such as the blood-brain barrier, and do not affect the integrity of the blood-brain barrier compared to AAV2. Conversely, AAV2 undergoes endocytosis and therefore transduces BMVECs with little diffusion across the endothelial barrier. The rational polyploids of the present invention participate in the transcytosis of BMVECs and transduce CNS regions, such as brain parenchymal cells. Primary BMVECs have served as a model for the human BBB to create an effective endothelial barrier and test AAV serotypes for transcytosis, endocytosis, and transduction, as described in J Neurochem 2017 January 140(2), 216-230; and J Neurochem 2017, 140, 192-194, both of which are incorporated herein by reference in their entirety.

[0194] Such haploid or polyploid virions are sometimes referred to as triploid virions, indicating that the capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes. Exemplary methods for producing such AAV haploid virions are described herein. By preventing translation of undesired open reading frames in VP3 from VP1 or VP2 AAV serotypes, these methods result in the production of a uniform population of resulting virions.

[0195] The ability to generate (e.g., substantially or completely) homogeneous populations of recombinant AAV haploid virions dramatically reduces or eliminates carryover of undesirable / contaminating virion properties (e.g., transduction specificity or antigenicity).

[0196] In one embodiment, the AAV haploid virions described herein that encapsidate the AAV genome (including the heterologous gene located between two AAV ITRs) can be formed with only two of the viral structural proteins, VP1 and VP3. In one embodiment, such AAV haploid virions are conformationally correct, i.e., have icosahedral symmetry with T=1. In one embodiment, the AAV haploid virions described herein are infectious. In one embodiment, the AAV haploid virions described herein have a different biodistribution compared to native AAV serotypes, e.g., the AAV haploid virions described herein have systemic transduction compared to native AAV serotypes. In one embodiment, the AAV haploid virions described herein have a reduced antigenic profile compared to native AAV serotypes, e.g., the AAV haploid virions described herein have a reduced ability to induce a humoral immune response or an increased ability to evade neutralizing antibodies (Nabs) compared to native AAV serotypes.

[0197] The AAV virion has a T=1 icosahedral symmetry and is composed of three structural viral proteins, VP1, VP2, and VP3. Approximately 60 copies of the three viral proteins, in a ratio of 1:1:8-10 (VP1:VP2:VP3, respectively), form the virion (Rayaprolu, V., et al., J. Virol. 87(24): 13150-13160 (2013)).

[0198] In one embodiment, an AAV virion encapsidating an AAV genome containing a heterologous gene located between two AAV ITRs can be formed with only two of the viral structural proteins, VP1 and VP3. In one embodiment, the virion is conformationally correct, i.e., has icosahedral symmetry with T=1. In one embodiment, the virion is infectious. Infectious virions contain VP1 / VP3 or VP1 / VP2 / VP3. Typically, virions containing only VP2 / VP3 or only VP3 are not infectious.

[0199] In some embodiments, the technology herein also provides an AAV capsid comprising a capsid protein VP1 from one or more first AAV serotypes and a capsid protein VP2 from one or more second AAV serotypes, wherein at least one of the first AAV serotypes, in any combination, is different from at least one of the second AAV serotypes, and at least one of VP1, VP2, or VP3 is from a rhesus AAV (AAVrh) serotype. In some embodiments, no chimeric viral structural proteins are present in the virion.

[0200] In some embodiments, the AAV haploid particles disclosed herein comprise a capsid protein comprising capsid protein VP3, wherein said capsid protein VP3 is from one or more third AAV serotypes, wherein said third AAV serotype is an AAV serotype that crosses the BBB, and wherein at least one of said one or more third AAV serotypes, in any combination, is different from said first AAV serotype and / or said second AAV serotype, and wherein either the first or second AAV serotype, but not both, is a rhesus AAV (AAVrh) serotype. In some embodiments, the AAV capsids described herein may comprise capsid protein VP1.5.

[0201] The present invention further provides an adeno-associated virus (AAV) particle comprising an AAV capsid, wherein the capsid comprises capsid protein VP1 from one or more first AAV serotypes (e.g., serotypes selected from Table 1) and capsid protein VP1.5 from one or more second AAV serotypes, wherein at least one of the first AAV serotypes, in any combination, is different from at least one of the second AAV serotypes, and wherein either the first AAV serotype or the second AAV serotype, but not both, is from rhesus AAV (AAVrh serotype).

[0202] In some embodiments, the capsid comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more third AAV serotypes that are AAV serotypes that cross the BBB, wherein at least one of said one or more third AAV serotypes is different from said first AAV serotype and / or said second AAV serotype, in any combination, and wherein at least one of the first AAV serotype, second AAV serotype, or third AAV serotype is from an AAV serotype that crosses the BBB, or in some embodiments, a non-human AAV serotype, such as a rhesus AAV (AAVrh) serotype, and the other serotype is selected from any AAV serotype selected from Table 1. In some embodiments, the AAV capsids described herein may comprise capsid protein VP1.5.

[0203] In some examples, the VP3 protein may be chemically modified to cross the BBB, or alternatively, in some embodiments, may include a targeting peptide to increase its ability to cross the BBB. The present invention further provides an adeno-associated virus (AAV) capsid comprising a capsid protein VP1 from one or more first AAV serotypes and a capsid protein VP1.5 from one or more second AAV serotypes, wherein at least one of the first AAV serotypes, in any combination, is different from at least one of the second AAV serotypes, and wherein either the first AAV serotype or the second AAV serotype, but not both, is from a rhesus AAV (AAVrh) serotype, and the other serotype is selected from any AAV serotype selected from Table 1.

[0204] In some embodiments, an AAV capsid of the invention comprises capsid protein VP3, wherein said capsid protein VP3 is from one or more third AAV serotypes that are AAV serotypes that cross the BBB, and at least one of said one or more third AAV serotypes is different from said first AAV serotype and / or said second AAV serotype, in any combination, and at least one of the first AAV serotype, second AAV serotype, or third AAV serotype is from an AAV serotype that crosses the BBB, and in some embodiments, a non-primate AAV, e.g., rhesus AAV (AAVrh), serotype; the other serotype may be selected from any AAV serotype selected from Table 1. In some embodiments, the AAV capsid protein described herein may comprise capsid protein VP2.

[0205] In some embodiments, the rational polyploid virions disclosed herein can be synthetic rational polyploid AAV viral vectors designed to display a variety of desirable phenotypes suitable for different in vitro and in vivo applications. Thus, in one embodiment, the present invention provides rational polyploid AAV viral vectors or virions. In one embodiment, the present invention provides a substantially pure population of rational polyploid AAV viral vectors or virions.

[0206] Infection of a host with a virus can stimulate the host's immune defense system to protect the infected host from the virus. One of the immune responses activated by a host to protect itself from foreign attack is a humoral immune response, which results in antibody-mediated immunity. In one embodiment, the rational polyploid virions disclosed herein elicit a reduced humoral response compared to the parent serotype and are less likely to be triggered by anti-AAV neutralizing antibodies compared to the parent serotype.

[0207] For the sake of explanation, neutralizing antibodies are distinct from non-neutralizing antibodies. Antibody-based immunity consists of neutralizing and non-neutralizing antibodies; non-neutralizing antibodies constitute a larger portion of the antibody pool generated during an immune response, but only a small portion are functional and participate in the clearance of infected cells, sometimes through interactions with other immune cells and / or the complement system. On the other hand, neutralizing antibodies specifically bind to epitopes critical for viral function and interfere with viral infectivity, e.g., block viral entry into host cells. Neutralizing antibodies (NAbs) bind to target cells and inhibit AAV transduction into target cells by several mechanisms. AAV neutralizing antibodies have been the focus of much research due to their significant detrimental effects on the efficacy of AAV-mediated gene therapy. Recent studies have shown that AAV-binding antibodies can also affect the distribution and safety of AAV vectors (Klasse et al., J Gen Virol, 2002, 83(Pt 9):2091; and Wang et al., Hum Gene Ther, 2011, 22(11):1389; the contents of each of these references are incorporated herein by reference in their entirety).

[0208] Detection of pre-existing neutralizing antibodies against AAV capsids and AAV serotypes in AAV gene delivery is essential for developing appropriate methods for overcoming the challenges posed by these pre-existing antibodies. This challenge can be overcome by using different / alternative AAV capsids and AAV serotypes in which lower titers or the absence of neutralizing antibodies are detected in patients or patient groups. III. Rational AAV polyploid vectors that cross the BBB

[0209] The present invention provides a number of synthetic, rational polyploid AAV viral vectors that exhibit a variety of desirable phenotypes suitable for different in vitro and in vivo applications. In particular, the present invention is based on the unexpected discovery that combining at least one VP3 structural protein, e.g., capsid protein, that efficiently crosses the BBB with at least one VP1 capsid protein and / or VP2 capsid protein from any different AAV serotype in individual capsids can result in improved AAV capsids, each of which possesses at least one property selected from the group consisting of increased tropism for the CNS and / or PNS, the ability to cross the blood-brain barrier (BBB) ​​and / or transduce the cerebrovascular (BBV) or blood components, enabling delivery of the rational AAV capsid to the brain via the cerebral circulation, eliciting a reduced humoral response, and the ability to escape neutralizing antibodies (Nabs) in vivo after administration. In some embodiments, one desirable feature of a rational polyploid AAV virion is its ability to be selected as a remedy vector. Such haploid or polyploid virions refer to capsid proteins VP1, VP2, and VP3 derived from at least two different serotypes. Exemplary methods for producing such rational polyploid AAV virions are described herein. By preventing the translation of undesired open reading frames of VP3 from AAV serotypes of VP1 and / or VP2 proteins, these methods result in the production of a uniform population of resulting virions.

[0210] The ability to generate a homogeneous (e.g., substantially or completely) population of recombinant rational polyploid AAV virions dramatically reduces or eliminates carryover of undesirable / contaminating virion properties (e.g., transduction specificity or antigenicity).

[0211] In some embodiments, a population of rational polyploid AAV virions disclosed herein exhibits enhanced transduction of one or more of endothelial cells, astrocytes, oligodendrocytes, CC1+ oligodendrocytes, neuronal subtypes (including NeuN+ cells throughout the brain, midbrain tyrosine hydroxylase (TH)+ dopaminergic neurons, and calbindin+ cerebellar Purkinje cells), interneuron populations, and CD31+ endothelial cells of the brain blood vessels (BBV) of a subject, compared to non-haploid AAV particles, e.g., AAV2, AAV5, or AAV8, that lack the ability to cross the blood-brain barrier. In some embodiments, a population of rational polyploid AAV virions exhibits comparable or enhanced transduction of one or more of the cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord of a subject, compared to non-haploid AAV particles that have the ability to cross the blood-brain barrier. In some aspects of the embodiment, comparable or enhanced transduction of a population of rational polyploid AAV virions into one or more of astrocytes, oligodendrocytes, CC1+ oligodendrocytes, neuronal subtypes (including NeuN+ cells throughout the brain, midbrain tyrosine hydroxylase (TH)+ dopaminergic neurons, calbindin+ cerebellar Purkinje cells), interneuron populations, and CD31+ endothelial cells of a subject compared to non-rational polyploid AAV particles capable of crossing the blood-brain barrier.

[0212] Methods for determining relative transduction efficiency are described in www.moleculartherapy.org vol. 19 no. 8, 1440-1448, 2011, which is incorporated herein by reference in its entirety. In some embodiments, the populations of haploid AAV virions disclosed herein exhibit enhanced transduction throughout the CNS compared to AAV2 or AAV5. In some embodiments, the population of haploid AAV virions exhibits enhanced transduction in the striatum compared to AAV2 or AAV5 virions, e.g., at least 20%, or 30%, or 40%, or 50%, 60%, 70%, 80%, 90%, or 95%, or 100% better transduction in the striatum compared to that of AAV2 or AAV5 virions under similar conditions, or at least 1.2-fold, or at least 1.5-fold, or at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or more enhanced transduction.

[0213] In one embodiment, the present invention provides an adeno-associated virus (AAV) haploid or polyploid capsid comprising a capsid protein VP1 from any serotype and at least one capsid protein VP3 from any AAV serotype that crosses the BBB and / or from a non-human primate, and which is not the same serotype as the VP1 (or, if present, VP2) AAV serotype. Preferably, such a population of rational polyploid virions is substantially homogeneous. In some embodiments, the rational polyploid virions disclosed herein may comprise a VP2 capsid protein, wherein the VP2 capsid protein is from any AAV serotype disclosed in Table 1 or a chimeric VP2 protein thereof, or the VP2 capsid protein is from any serotype that is the same as the serotype from which VP3 is derived, or alternatively, the VP2 capsid protein is from a serotype different from the serotype from which VP3 is derived. Exemplary VP1-VP2-VP3 configurations of rational polyploid, e.g., haploid virions disclosed herein may be represented as XYZ, XXZ, ZXZ, XZZ, where X, Y, and Z are each selected from only one AAV serotype and are different serotypes; X and Z can be selected from any serotype disclosed in Table 1; and Z is from any serotype that crosses the BBB. That is, in any rational haploid vector, VP3 is from only one Z serotype, VP1 is from only one X serotype, etc. In some embodiments, X and Y can be selected from any serotype that crosses the BBB and / or a non-human primate AAV serotype, although in such instances they are from a different serotype than the serotype of Z. In some embodiments, Z is from any non-primate AAV serotype, e.g., a rhesus serotype. In some embodiments, Z may be selected from any of the serotypes AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43.

[0214] In one embodiment, the rational haploid virions described herein that encapsidate an AAV genome (including a heterologous gene located between two AAV ITRs) can be formed with only two of the viral structural proteins, VP1 and VP3. In one embodiment, such rational haploid virions are conformationally correct, i.e., have icosahedral symmetry with T=1. In one embodiment, the rational haploid AAV virions described herein are infectious.

[0215] The AAV virion has a T=1 icosahedral symmetry and is composed of three structural viral proteins, VP1, VP2, and VP3. Approximately 60 copies of the three viral proteins, in a ratio of 1:1:8-10 (VP1:VP2:VP3, respectively), form the virion (Rayaprolu, V., et al., J. Virol. 87(24): 13150-13160 (2013)).

[0216] In one embodiment, a rational polyploid AAV virion is an isolated virion having at least one viral structural protein, VP1, VP2, and VP3, from a different serotype than the other VPs, and each VP is from only one serotype. Simply for illustration, if VP1 is from only AAV2, VP2 is from only AAV4, and VP3 is from a BBB-crossing serotype that is neither AAV2 nor AAV4. In some embodiments, a population of rational polyploid AAV virions has improved biodistribution in the brain and spinal cord compared to AAV8. In some embodiments, the CNS biodistribution is at least 0.1 vg / cell, at least 0.2 vg / cell, at least 0.4 vg / cell, at least 0.6 vg / cell, at least 0.8 vg / cell, at least 1 vg / cell, at least 2 vg / cell, at least 3 vg / cell, at least 4 vg / cell, at least 5 vg / cell, at least 6 vg / cell, at least 7 vg / cell, at least 8 vg / cell, at least 9 vg / cell, at least 10 vg / cell, at least Between 10-15 vg / cell, at least 15-20 vg / cell, at least 20-30 vg / cell, at least 30-40 vg / cell, at least 40-50 vg / cell, at least 50 vg / cell, at least 50-60 vg / cell, at least 60-70 vg / cell, at least 70-80 vg / cell, at least 80-90 vg / cell, at least 90-100 vg / cell, at least 100 vg / cell, or more.

[0217] CNS biodistribution corresponds to biodistribution in brain and spinal cord regions. Non-limiting exemplary CNS biodistribution regions include the olfactory bulb, striatum, hippocampus, cortex, thalamus, hypothalamus, cerebellum, medulla, cervical plexus, thoracic plexus, lumbar plexus, choroid plexus, habenula, horn of Ammon, dentate gyrus, caudate putamen, and amygdala. In some embodiments, a population of rational polyploid AAV virions exhibits enhanced transduction in neurons over glial cells. In some embodiments, a population of rational polyploid virions exhibits enhanced transduction in astrocytes. In some embodiments, a population of rational polyploid AAV virions exhibits significant transduction in tissues other than the CNS. In some aspects of the embodiments, a population of rational polyploid AAV virions exhibits transduction in the lung and kidney, and has supraphysiological levels in the liver, heart, skeletal muscle, intestine, and spleen.

[0218] In some embodiments, populations of rational polyploid AAV virions of the present invention exhibit significant transduction of endothelial cells of the cerebrovascular (BBV). In some embodiments, populations of rational polyploid AAV virions of the present invention exhibit significant transduction of cells that are part of the BBB, including any one or more of the endothelial cells of the BBB and the cell surface processes of astrocytes called astropods (also known as "glial limiting membranes") that surround the endothelial cells of the BBB. In some embodiments, populations of rational polyploid AAV virions of the present invention exhibit significant transduction of blood components that can cross the BBB, including, but not limited to, activated T cells, blood monocytes, and macrophages.

[0219] In one embodiment, a rational polyploid AAV virion encapsidating an AAV genome containing a heterologous gene between two AAV ITRs can be formed with only two of the viral structural proteins, VP1 and VP3, where VP3 is from a serotype that efficiently crosses the BBB. In one embodiment, the virion is conformationally correct, i.e., has icosahedral symmetry with T=1. In one embodiment, the virion is infectious.

[0220] Infectious virions contain VP1 / VP3 or VP1 / VP2 / VP3. Typically, virions containing only VP2 / VP3 or only VP3 are not infectious.

[0221] In some embodiments, the viral structural protein VP2 used to generate these populations of rational polyploid AAV virions can be from any of the 12 serotypes of AAV isolated for gene therapy, other species, mutant serotypes, genetically shuffled serotypes of such, e.g., AAV1, AAV2, AAV VP1.5, AAV4 VP2, AAV4 VP3, AAV Rh10 VP3, AAV Rh74 VP3, AAV Rh74 VP2, or any other AAV serotype desired, e.g., as disclosed in Table 1.

[0222] As disclosed herein, the VP3 structural capsid protein is selected from any AAV serotype that efficiently crosses the BBB, such AAV serotypes being selected from among AAV1, AAV6, AAV6.2, AAV7, AAV9, rh10, rh74, rh39, and rh43. Exemplary rational polyploid vectors comprising VP1, VP2, and VP3 proteins for use in the methods and compositions disclosed herein include, but are not limited to, AAV-XY-1, AAV-XX-1, AAV-XY-6, AAV-XX-6, AAV-XY-6.2, AAV-XX-6.2, AAV-XY-7, AAV-XX-7, AAV-XY-9, AAV-XX-9, AAV-XY-rh10, AAV-XX-rh10, AAV-XY-rh74, AAV-XX-rh74, AAV-XY-rh39, AAV-XX-rh39, AAV-XY-43, AAV-XX-43, where X and Z are each selected from only one AAV serotype and are different serotypes and are selected from any of the serotypes disclosed in Table 1. In some embodiments, X and / or Y can be from any serotype that crosses the BBB, or alternatively, can be from any non-human primate AAV serotype, where the serotype of X and / or Y, if present, is different from that of VP3. In some embodiments where a rational polyploid comprises VP1, VP2, and VP3 proteins, the serotype of the VP1 protein serotype is the same as the serotype of the VP3 protein that crosses the BBB, and the serotype of the VP2 protein is from a different serotype. In some embodiments where a rational polyploid comprises VP1, VP2, and VP3 proteins, the serotype of the VP2 protein serotype is the same as the serotype of the VP3 protein that crosses the BBB, and the serotype of the VP1 protein is from a different serotype.

[0223] In some embodiments, a rational polyploid vector may comprise only VP1 and VP3 proteins, or only VP2 and VP3 proteins; exemplary such rational polyploid vectors for use in the methods and compositions disclosed herein include, but are not limited to, AAV-X-1, AAV-X-6, AAV-X-6.2, AAV-X-7, AAV-X-9, AAV-X-rh10, AAV-X-rh74, AAV-X-rh39, AAV-X-43, where X is either VP1 or VP2 and is selected from only one serotype selected from any of the serotypes disclosed in Table 1. In some embodiments, X may be from any serotype that crosses the BBB, or alternatively, may be from any non-human primate AAV serotype, where the serotype of X, if present, is different from the AAV serotype of VP3.

[0224] In some embodiments, the VP3 can be selected from any AAV serotype that crosses the BBB, including, but not limited to, AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrhlO, AAVrh74, AAVrh39, and AAVrh43, or variants having at least 85% amino acid sequence identity to the native amino acid sequence. AAV serotypes that cross the BBB are disclosed in Zhang et al., Mol. Therapy, 19(8); 2011; 1440-1448; Nonnenmacher et al., Mol. Ther: Methods and Clinical development; 2021, 336; and Gao et al., J. Virol, 2004; 6381-6388, which are incorporated herein by reference in their entireties.

[0225] In some embodiments, the rational AAV polyploid vectors disclosed herein comprise VP1, VP2, or VP3 proteins, wherein VP1, VP2, or VP3 are each from a different serotype, and serotype X or Y is any AAV serotype selected from Table 1, and serotype Z is from any serotype that crosses the BBB only, and / or alternatively, from a non-human primate AAV that crosses the BBB but is not serotype X or serotype Y, or any serotype or chimeric or non-naturally occurring serotype.

[0226] In some embodiments, the AAV rational polyploid comprises three serotypes, e.g., X, Y, and Z, where VP1, VP2, and VP3 are from only one serotype, each of which is a different serotype, and X and Y can be selected from any serotype selected from Table 1, and VP3 (serotype Z) is from any AAV serotype that crosses the BBB. Such combinations are shown in Table 2.

[0227] Table 2: Table of different capsid protein combinations of rational AAV polyploids, including three different serotypes: X, Y, and Z (where serotype Z is selected from any AAV serotype that crosses the BBB). [Table 2]

[0228] In some embodiments, the AAV rational haploid vector comprises only two serotypes, one of which is any AAV serotype that crosses the BBB (referred to as serotype Z), and one of which (serotype X) is from any serotype selected from Table 1. In some embodiments, exemplary serotypes of serotype A include, but are not limited to, AAV8, AAV9, AAV3, and AAV3b. In some embodiments, exemplary serotypes of serotype Z are AAV serotypes that cross the BBB selected from any of AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrhlO, AAVrhl74, and AAVrhl9. In some embodiments, the serotype Z serotype may also be selected from any non-primate AAV serotype, including rhesus serotypes AAVrh.10, AAVrh.74, AAVrh.73, AAVrh.75, AAVrh.76, rAAVrh39, rAAVrh.43. IV. Exemplary AAV8 Haploid Vectors that Cross the BBB

[0229] In one embodiment, the rational polyploid vector disclosed herein is an adeno-associated virus 8 (AAV8) haploid or polyploid capsid, wherein the capsid comprises a capsid protein VP1 from the AAV8 serotype and at least one capsid protein VP3 from any AAV serotype that crosses the BBB and / or from a non-human primate, but which is not the AAV8 serotype. Preferably, such a population of AAV8 haploid virions is substantially homogeneous. In some embodiments, the AAV8 haploid capsid of the present invention may comprise a VP2 capsid protein, wherein the VP2 capsid protein is from the AAV8 serotype or a chimeric VP2 protein thereof, or the VP2 capsid protein is from any serotype that is the same as the serotype from which VP3 is derived, or alternatively, the VP2 capsid protein is from a serotype different from the serotype from which VP3 is derived. Exemplary arrangements of VP1-VP2-VP3 in the AAV8 haploid capsids of the invention may be represented as AAV8-8-Y, AAV8-XY, AAV8-YY, where X is a VP2 capsid protein from any serotype (except AAV8), Y is a VP3 protein from any serotype (except AAV8), X and Y are from different serotypes, and X and Y can be selected from any serotype that crosses the BBB and / or non-human primate AAV serotypes.

[0230] In some embodiments, AAV8 haploid or polyploid virions as disclosed herein are produced that contain VP1 from an AAV8 serotype and at least one VP3 capsid protein, where VP3 is not from AAV8 but is selected from any serotype that crosses the BBB and / or is a non-human primate AAV serotype. For example, AAV8 haploid or polyploid virions are produced in which VP1 and optionally VP2 are from the AAV8 serotype, and VP3 is not from AAV8 but is selected from any AAV serotype that crosses the BBB and / or is a non-human primate AAV serotype.

[0231] In one embodiment, the AAV8 virion is an isolated virion having at least VP1 from the AAV8 serotype and one of the viral structural proteins, VP2 and / or VP3, from a serotype different from AAV8, where either VP2 and / or VP3 is from any AAV serotype whose native AAV vector crosses the BBB. For example, the AAV8 haploid virion described herein can contain VP1 from AAV8 and VP2 or VP3, or both VP2 and VP3, from AAV9, AAV7 (accession number AF513852, which is the entire genome of AAV7), rAAVrh74, rAAVrh.39, rAAVrh.43, or variants thereof.

[0232] In one embodiment, the AAV8 virion is an isolated virion having at least VP1 from the AAV8 serotype and one of the viral structural proteins, VP2 and / or VP3, from a serotype different from AAV8, where either VP2 and / or VP3 is from any non-human primate AAV serotype. For example, VP1 is from AAV8 only, and VP2 and / or VP3 are from rhesus macaques. For example, the AAV8 haploid virion described herein can contain VP1 from AAV8 and VP2 or VP3, or both VP2 and VP3, from rAAVrh.10, rAAVrh.74, rAAVrh.39, or rAAVrh.43, or variants thereof. Throughout this application, the virions described herein may be referred to as AAV(VP1)-VP2-VP3, or alternatively as "AAVnnn." For example, for illustrative purposes only, a virion having VP1 from AAV8, VP2 from AAV8, and VP3 from AAVrh.10 may be designated as AAV8-8-rh10 or AAV88rh10. Thus, using this nomenclature, exemplary AAV8 haploid virions herein include, but are not limited to, AAV8-8-rh10, AAV8-8-rh74, AAV8-8-rh39, AAV8-8-rh43, or variants thereof, where the VP1 and VP2 capsid proteins are from AAV8 and the VP3 is from the indicated serotype. Other exemplary AAV8 haploid vectors include, but are not limited to, AAV8-X-rh10, AAV8-X-rh74, AAV8-X-rh39, AAV8-X-rh43, or variants thereof, where X is VP2 from any serotype except AAV8. In some embodiments, X is a VP2 capsid protein from any rhesus AAV serotype, including but not limited to AAVrhlO, rAAVrh74, rAAVrh.39, or rAAVrh.43, or a variant thereof.

[0233] In some embodiments herein, virion particles can be constructed in which the VP1 capsid protein is from AAV8 and at least one of the VP2 or VP3 viral proteins is not from AAV8. In some embodiments, VP1 and VP2 can be from the AAV8 serotype. In all embodiments disclosed herein, VP3 is not from AAV8. In some embodiments, VP2 and VP3 are from the same serotype, e.g., AAV8-rh10-rh10. In some embodiments, VP2 and VP3 are from different serotypes, e.g., AAV8-8-rh10 or AAV8-rh74-rh10. In each aspect of all embodiments disclosed herein, at least VP1 from AAV8 and VP3 capsid protein from another serotype are required to form virion particles capable of encapsidating an AAV genome. For each virion particle, the capsid proteins (VP1, VP2, and / or VP3) are of the same type (e.g., all virions contain AAV8 VP1). In some embodiments, at least one of the viral capsid proteins is a chimeric viral protein, and at least one of the other two viral proteins is not chimeric. In some embodiments, VP1 is a chimeric AAV8 VP1 protein. In some embodiments, VP1 and VP2 are chimeric, and only VP3 is non-chimeric. For example, only viral particles are composed of VP1 / VP2 from a chimeric AAV2 / 8 (AAV2 N-terminus and AAV8 C-terminus) paired with only VP3 from any other non-AAV8 vector, such as rh10, rh74, etc. In another embodiment, only VP3 is chimeric, and VP1 and VP2 are non-chimeric. In another embodiment, at least one of the viral proteins is from an entirely different serotype. In another example, none are chimeric.

[0234] In some embodiments, the rational polyploid virions disclosed herein are AAV8 haploid virions and may be selected from any of AAV88rh10, AAV88rh74, AAV88rh74vv, AAV88rh10LP2, AAV8rh74LP2, and AAV88rh74vvLP2, each of which is described in the Examples section provided herein. In some embodiments, these AAV8 haploid virions may be designated as AAV8-8-rh10, AAV8-8-rh74, AAV8-8-rh74vv, AAV8-8-rh10LP2, AAV8-8-rh74LP2, and AAV8-8-rh74vvLP2. Simply by way of illustration, these AAV haploid virions comprise AAV8 VP1 and VP2 structural proteins (e.g., SEQ ID NOs: 7 and 8), or comprise a modified protein of SEQ ID NO: 7 or 8 and a VP3 protein selected from any of rh10 VP3 (SEQ ID NO: 1), rh74 VP3 (SEQ ID NO: 3), rh74vv VP3 (SEQ ID NO: 2), rh10-LP2 VP3 protein (SEQ ID NO: 14), rh74-LP2 VP3 protein (SEQ ID NO: 17), and rh74vv-LP2 VP3 protein (SEQ ID NO: 15), or a VP3 protein having an amino acid sequence that is at least 85% sequence identity to any of SEQ ID NOs: 1, 2, 3, 14, 15, and 18. In some embodiments, the modified AAV8 VP1 or VP2 protein comprises an insertion of a peptide, including, but not limited to, a targeting peptide or a BBB-penetrating peptide disclosed herein, at an appropriate site in the VP1 and / or VP2 protein. In some embodiments, the linker, e.g., a peptide linker, may flank the targeting peptide (i.e., each end of the targeting peptide may constitute a peptide linker), or there is a peptide linker located at one end of the targeting peptide.In some embodiments, the AAV8 VP1 and / or VP2 structural proteins are modified as a double mutant (Y444+733F) or triple mutant (Y444+733F T494V) as disclosed in Gilkes Site-specific modifications to AAV8 capsid yields enhanced brain transduction in the neonatal MPS IIIB mouse. Gene Ther (2020), which is incorporated herein by reference in its entirety;

[0235] In some embodiments, only virions containing the VP1 capsid protein from AAV8 and the VP3 capsid protein from a serotype other than AAV8 are produced. For example, VP1 and VP2 are from the AAV8 serotype, and VP3 is from an alternative serotype only. In other embodiments, VP1 is from the AAV8 serotype, VP2 and VP3 are from another serotype only, and the VP2 and VP3 capsid proteins are from a serotype that crosses the BBB and / or is a non-human primate AAV serotype. In another embodiment, only particles are produced in which VP1 is from the AAV8 serotype, VP2 is from a second serotype, and VP3 is from yet another serotype, referred to herein as polyploid AAV8 virions.

[0236] This can be done, for example, by site-specific deletions and / or additions that alter splice donor sites, splice acceptor sites, start codons, and combinations thereof.

[0237] This allows for a method for producing a population that is a substantially homogeneous population of polyploid virions, eg, haploid particles.

[0238] In some embodiments, exemplary AAV8 haploid vectors are selected from the following: a. AAV8-8-rhY (where Y is VP3 from a non-human primate AAV serotype, such as rhesus macaque); b. AAV8-8-rh10; c. AAV8-X-rh10, where x is any serotype except Rh10; d. AAV8-X-rh10, where x is any serotype except AAV8; e. AAV8-8-rh74; f. AAV8-8-rh74vv, in which the rh74 VP3 capsid protein contains the W581VV modification; g. AAV8-X-rh74 or rh74vv, where x is any serotype except Rh74; h. AAV8-X-rh74 / rh74vv, where x is any serotype except AAV8; i. AAV8-8-Y, where Y is a VP3 capsid protein from any serotype that crosses the BBB (e.g., exemplary VP3 capsid proteins that cross the BBB can be selected from AAV1, AAV6, AAV6.2, AAV7 (accession number AF513852, which is the entire genome of AAV7), AAV9, rAAVrh10, rAAVrh74, rAAVrh39, rAAVrh43); j. AAV8-XY, where X is any serotype except AAV8, and Y is VP3 from any serotype that crosses the BBB, and Y is not from AAV8; k. AAV8-XY, where X is any serotype that crosses the BBB and Y is VP3 from any serotype that crosses the BBB, and X and Y are not the VP2 or VP3 capsid proteins from the AAV8 serotype, respectively, and the BBB-crossing serotype can be selected from AAV1, AAV6, AAV6.2, AAV7 (accession number AF513852, which is the entire genome of AAV7), AAV9, rAAVrhlO, rAAVrh74, rAAVrh39, and rAAVrh43; l. AAV8-XX, where X is any serotype that crosses the BBB, and X is not the VP2 or VP3 capsid protein, respectively, from the AAV8 serotype, and the BBB-crossing serotype can be selected from AAV1, AAV6, AAV6.2, AAV7 (accession number AF513852, which is the entire genome of AAV7), AAV9, rAAVrhlO, rAAVrh74, rAAVrh39, and rAAVrh43; m. AAV8-XY, where X is any serotype except AAV8, and Y is VP3 from any serotype that crosses the BBB, and Y is not from AAV8.

[0239] Exemplary AAV haploid vectors that cross the BBB in some embodiments are disclosed in Table 4.

[0240] Table 4: Exemplary rational polyploid or polyploid vectors in which VP3 is from a serotype that crosses the BBB (e.g., selected from AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrhlO, AAVrh74, AAVrh39), and VP1 and / or VP2 are from a serotype different from that of VP3 and may also be from a serotype that crosses the BBB, or may be selected from AAV8 or X, where X is a serotype that is not AAV8 and may be selected from any serotype from Table 1. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] (i) AAV8-8-rh10 haploid vector

[0241] In some embodiments, an exemplary AAV haploid vector is AAV8-8-rh10, in which VP1 and VP2 are exclusively from the AAV8 serotype and VP3 is exclusively from the rhesus AAV10 (AAVrh10) serotype. In some embodiments, the VP3 capsid protein is a chimeric VP3 protein from the AAVrh10 serotype. In some embodiments, the AAV8-8-rh10 haploid vector comprises a VP3 capsid protein having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO: 1, where SEQ ID NO: 1 is the amino acid sequence of the codon-optimized VP3 capsid protein from AAVrh10. In some embodiments, the VP3 is a modified VP3 protein containing at least one, or at least two, or at least three modifications selected from Q214N, S462N, and D517E of SEQ ID NO: 1. SEQ ID NO:1, which comprises the amino acids of VP3 from the AAVrh10 serotype, is encoded by the nucleic acid sequence of SEQ ID NO:5, or a variant having at least 95% or at least 98% nucleic acid sequence identity to SEQ ID NO:5.

[0242] In some embodiments, an exemplary AAV8 haploid vector is AAV8-8-rh10, in which VP1 and VP2 are encoded by the nucleic acid sequence of SEQ ID NO:6, which nucleic acid sequence includes M203V and M211V, M204 and / or M212, M204 and / or M212, such that the VP3 protein from the AAV8 serotype is not expressed. In some embodiments, the nucleic acid variant includes modification of at least one base of the ACG at positions 412-414 of SEQ ID NO:6 to destroy or render inoperable the start codon (threonine (T or Thr)) of VP2; thus, this nucleic acid of SEQ ID NO:6 encodes only VP1 of the AAV8 serotype, and neither VP2 nor VP3 from the AAV8 serotype. In such embodiments, the AAV8 haploid vector may comprise a VP2 protein from a different serotype, e.g., an AAVrh serotype, or alternatively, VP2 may be absent from an AAV8 haplotype as discussed herein, and thus, a VP3 protein from an AAV8 serotype is not expressed. In some embodiments, the nucleic acid variant comprises a modification of at least one base of the ACG at positions 412-414 of SEQ ID NO: 6 to destroy or render inoperable the start codon of VP2 (threonine (T) or Thr), and thus, this nucleic acid of SEQ ID NO: 6 encodes only VP1 of the AAV8 serotype, and neither VP2 nor VP3 from the AAV8 serotype. In such embodiments, the AAV8 haploid vector may comprise a VP2 protein from a different serotype, e.g., an AAVrh serotype, or alternatively, VP2 may be absent from an AAV8 haplotype as discussed herein.

[0243] As shown in the Examples herein, the rational polyploid AAV8-8-rh10 elicited a reduced humoral response compared to the parental AAV8 (Figure 25A), demonstrating that this AAV8-8-rh10 haploid has a distinct, reduced antigenic profile compared to the parental AAV8 vector. Figures 8B and 10C also demonstrate that the production yield and specific productivity from AAV8-8-rh10 were comparable to the AAV8 or AAVrh10 controls.

[0244] In some embodiments, AAV8-8-rh10 haploids were produced using the plasmid of SEQ ID NO: 12, which contains the construct of SEQ ID NO: 13. (ii) AAV8-8-rh74 haploid vector

[0245] In some embodiments, an exemplary AAV haploid vector is AAV8-8-rh74, in which VP1 and VP2 are exclusively from the AAV8 serotype and VP3 is exclusively from the rhesus AAV74 (AAVrh74) serotype. In some embodiments, the VP3 capsid protein is a chimeric VP3 protein from the AAVrh74 serotype. In some embodiments, the AAV8-8-rh74 haploid vector comprises a VP3 capsid protein having the amino acid sequence of SEQ ID NO:3, or an amino acid sequence with at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:3, where SEQ ID NO:3 is the amino acid sequence of the wild-type VP3 capsid protein from AAVrh74.

[0246] In some embodiments, the rh74 VP3 is a modified VP3 protein comprising at least one or more amino acid modifications. In specific embodiments, the AAVrh74 VP3 capsid protein is a modified VP3 protein comprising the W581VV modification, in which two consecutive valine (V or val) amino acids are substituted with tryptophan (W or Trp) at amino acid position 581 of SEQ ID NO: 3 (using nomenclature / numbering from the amino acid sequence of the VP1 capsid protein from AAVrh74). Thus, in some embodiments, the AAV haploid vector is an AAV8-8-rh74vv haploid vector comprising a VP3 capsid protein having the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence with at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO: 2, wherein SEQ ID NO: 2 is the amino acid sequence of the rh74vv-VP3 capsid protein comprising the W581VV modification.

[0247] SEQ ID NO:2, which contains the amino acid sequence of the rh74vv-VP3 capsid protein, is encoded by the nucleic acid sequence of SEQ ID NO:4.

[0248] In some embodiments, the AAV haploid vector is an AAV8-8-rh74vv haploid vector comprising a VP3 capsid protein having the amino acid sequence of SEQ ID NO:2, or an amino acid sequence with at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:2, wherein SEQ ID NO:2 is the amino acid sequence of the rh74vv-VP3 capsid protein comprising the W581VV modification, and the rh74vv-VP3 capsid protein is encoded by a nucleic acid sequence comprising SEQ ID NO:4, or a nucleic acid sequence with at least 85%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 95%, or at least 98% sequence identity to SEQ ID NO:4.

[0249] In one embodiment of any aspect herein, the AAVrh74 VP3 protein in the AAV8-8-rh74vv haploid vector has the amino acid sequence of SEQ ID NO:2, or the amino acid sequence of a protein having at least 85% sequence identity to SEQ ID NO:2, or the mutant AAVrh74 VP3 includes at least one of the following modifications of SEQ ID NO:2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P.

[0250] In one embodiment of any aspect herein, the AAVrh10 VP3 protein in the AAV8-8-rh74vv haploid vector is encoded by the nucleic acid of SEQ ID NO: 5, which includes at least one or more of Q214N, S462N, and D517E mutations compared to the AAVrh10_VP3 nucleic acid of SEQ ID NO: 5, or is encoded by the nucleic acid of SEQ ID NO: 5, which includes a nucleic acid sequence with at least 85% sequence identity to SEQ ID NO: 5, which includes at least one mutation selected from Q214N, S462N, and D517E.

[0251] In one embodiment of any aspect herein, the AAVrh74 VP3 protein in the AAV8-8-rh74vv haploid vector comprises the amino acid sequence of SEQ ID NO: 2 or 3, or the amino acid sequence of a protein having at least 85% sequence identity to SEQ ID NO: 2 or 2, or comprises at least one of the following amino acid modifications of SEQ ID NO: 2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P.

[0252] Importantly, as disclosed herein in the Examples and in Figures 30B-30C, the haploid AAV8-8-rh74 vector has improved brain and spinal cord transduction and BBB crossing capabilities compared to mice immunized with the parental AAV serotype.

[0253] Importantly, as disclosed in the Examples herein, the haploid AAV8-8-rh74 vector has an improved ability to escape neutralizing antibodies from sera immunized with the parental serotype. Importantly, AAV8-8-rh74 is surprisingly more efficient than the parental AAV8 or AAVrh10 at transducing the entire body of mice after systemic administration, and more efficient than the haploid AAV8-8-rh10 (see Figures 21A-21D). Furthermore, AAV8-8-rh74, in contrast to AAV8 and AAVrh10, exhibited the ability to cross the blood-brain barrier, thus demonstrating unexpected results compared to its parental AAV8 and a different phenotype from that exhibited by the AAV8 haploid. Furthermore, the haploid vector AAV8-8-rh74 transduced Pro10 cells similarly to the AAV8 control and, importantly, transduced GM16095 cells significantly more efficiently than the parental AAV8 vector. Furthermore, the AAV8-8-rh74 haploid was found to evade anti-AAV8 neutralizing antibodies (see Figures 18A-18B, Figure 19) and generate a reduced humoral response compared to the parental AAV8 (Figure 25A), thus demonstrating that this AAV8-8-rh74 haploid has a distinct, reduced antigenic profile compared to the parental AAV8 vector.

[0254] In some embodiments, the AAV8-8-rh74 haploid was produced using the plasmid of SEQ ID NO: 10, which contains the construct of SEQ ID NO: 11. V. AAV Haploid or Polyploid Capsid Variants

[0255] In some embodiments, the AAV haploid or polyploid virions encompassed herein may be formed by more than the three typical viral structural proteins, VP1, VP2, and VP3 (see, for example, Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such AAV haploid or polyploid viral capsids are also encompassed by the present invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV haploid or polyploid virion that encapsidates an AAV genome, wherein VP1 is from any AAV serotype listed in Table 1, and VP3 is from any serotype that crosses the BBB. In a further embodiment, the isolated AAV haploid or polyploid virion has at least two viral structural proteins from the group consisting of AAV capsid proteins, VP1, VP1.5, and VP3, sufficient to form an AAV haploid or polyploid that encapsidates an AAV genome, wherein VP1 or VP1.5 is from any AAV serotype listed in Table 1, and VP3 is from any serotype that crosses the BBB. For example, VP1.5 can be from any AAV serotype listed in Table 1, and VP3 can be from any one or more of the AAV serotypes that cross the BBB, including, but not limited to, AAV1, AAV6, AAV6.2, AAV7, AAV9, rAAVrhlO, rAAVrh74, rAAVrh39, and rAAVrh43.

[0256] In some embodiments, a capsid of the invention comprises capsid protein VP1.5, which is not from the same serotype as VP1 or the VP3 capsid protein. In some embodiments, an AAV haploid or polyploid capsid protein described herein can comprise capsid protein VP2, as described herein.

[0257] In some embodiments, capsids of the invention comprise capsid protein VP2, which is selected from the AAV8 serotype or any serotype listed in Table 1, or the same serotype as the VP3 protein of the capsid, or a different serotype than that used for VP3. In some embodiments, the rational polyploid AAV vectors described herein can comprise capsid protein VP1.5. VP1.5 is described in U.S. Patent Application Publication No. 2014 / 0037585, and the amino acid sequence of VP1.5 is provided herein.

[0258] Thus, in certain embodiments, at least one of the viral structural proteins, e.g., VP1, VP2, or VP3, can be a chimeric viral structural protein, i.e., contain segments from more than one protein. In one embodiment, the chimeric viral structural proteins are all from the same serotype. In another embodiment, the chimeric viral structural proteins are composed of components from more than one serotype, but these serotypes are different from at least one other serotype. In one embodiment, the viral structural proteins are not chimeric. In one embodiment, the chimeric AAV structural proteins do not contain structural amino acids from canine parvovirus. In one embodiment, the chimeric AAV structural proteins do not contain structural amino acids from b19 parvovirus. In one embodiment, the chimeric AAV structural proteins do not contain structural amino acids from canine parvovirus or b19 parvovirus. In one embodiment, the chimeric AAV structural proteins contain only structural amino acids from AAV. In some embodiments, the rational polyploid AAV vector does not contain a chimeric VP1 protein. In some embodiments, the rational polyploid AAV vector comprises a chimeric VP1 protein, for example, a chimeric VP1 protein from the AAV8 serotype.

[0259] In some embodiments, the rational polyploid AAV virions disclosed herein may be formed by more than the three typical viral structural proteins, VP1, VP2, and VP3 (see, for example, Wang, Q. et al., "Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors," Molecular Therapy: Methods and Clinical Development, Vol. 4, 149-158 (2017)). Such viral capsids are also encompassed by the present invention. For example, an isolated AAV virion having sufficient viral capsid structural proteins to form an AAV virion that encapsidates an AAV genome, wherein at least one of the viral capsid structural proteins is different from the other viral capsid structural proteins, and each viral capsid structural protein is of the same type only. In a further embodiment, the isolated AAV virion has at least two viral structural proteins selected from the group consisting of AAV capsid proteins VP1, VP2, VP1.5, and VP3, wherein the two viral proteins are sufficient to form an AAV virion that encapsidates the AAV genome, and at least one of the viral structural proteins present is from a different serotype than the other viral structural proteins, such that VP1 is from only one serotype, VP2 is from only one serotype, and VP3 is from only one serotype that efficiently crosses the BBB. In one embodiment, the rational polyploid comprises VP1.5. By way of example only, VP1.5 can be from AAV serotype 8, and VP3 can be from an AAV serotype that crosses the BBB and / or alternatively, from an AAVrh serotype.

[0260] In some embodiments, only virions are produced that contain a VP1 capsid protein from one serotype (e.g., a serotype listed in Table 1) and a VP3 capsid protein from a serotype different from that of VP1, and in which the AAV serotype efficiently crosses the BBB. For example, VP1 and VP2 are from any serotype listed in Table 1, e.g., the AAV8 serotype, and VP3 is from only the alternative serotype that crosses the BBB. In other embodiments, VP1 is from only one serotype (e.g., the AAV8 serotype), VP2 and VP3 are from only another serotype, and the VP2 and VP3 capsid proteins are from a serotype that crosses the BBB. In another embodiment, only particles are produced in which VP1 is from a serotype listed in Table 1 (e.g., the AAV8 serotype), VP2 is from only the second serotype, and VP3 is from yet another serotype that crosses the BBB, referred to herein as polyploid AAV8 virions.

[0261] This can be done, for example, by site-specific deletions and / or additions that alter splice donor sites, splice acceptor sites, start codons, and combinations thereof.

[0262] This allows for a method for producing a population that is a substantially homogeneous population of polyploid virions, eg, haploid particles.

[0263] In some embodiments, the technology herein also provides an AAV capsid comprising a capsid protein VP1 from one or more first AAV serotypes and a capsid protein VP2 from one or more second AAV serotypes, wherein at least one of the first AAV serotypes, in any combination, is different from at least one of the second AAV serotypes, and at least one of VP1, VP2, or VP3 is from a serotype that crosses the BBB, or alternatively, is from a non-primate AAV serotype, such as a rhesus AAV (AAVrh) serotype. In some embodiments, no chimeric viral structural proteins are present in the virion. VI. Modifications to the VP1 and VP3 Capsid Proteins

[0264] The present invention further provides compositions, which may be pharmaceutical formulations, comprising the capsid protein, capsid, viral vector, AAV particle compositions and / or pharmaceutical formulations of the invention and a pharmaceutically acceptable carrier.

[0265] In some non-limiting examples, the present invention provides AAV capsid proteins (VP1, VP1.5, VP2, and / or VP3) comprising modifications of the amino acid sequence in triad loop 4 (Opie et al., J. Viral. 77: 6995-7006 (2003)), as well as viral capsids and viral vectors comprising the modified AAV capsid proteins. The rational polyploid or polyploid AAV vectors disclosed herein comprising at least one VP structural protein from an AAV serotype that crosses the BBB, e.g., VP3 from a serotype that crosses the BBB, have a distinct transduction profile compared to the parent AAV vector after systemic or intrathecal delivery, and surprisingly, have an increased ability to cross the BBB after intrathecal or systemic delivery, and / or an increased ability to transduce endothelial cells of the BBB and / or blood cells that cross the BBB. For example, the AAV8-8-rh74 haploid AAV vector disclosed in the Examples herein exhibits significantly increased systemic transduction after systemic administration, thereby increasing transduction of each target tissue, e.g., skeletal muscle, cardiac muscle, etc.

[0266] In some embodiments, the modified AAV capsid may be composed of VP1, VP2, and / or VP3 generated by DNA shuffling to generate cell-type-specific vectors by directed evolution. DNA shuffling for AAV is generally described in Li, W. et al., Mol. Ther. 16(7): 1252-12260 (2008), which is incorporated herein by reference. In some embodiments, DNA shuffling may be used to generate VP1, VP2, and / or VP3 using capsid gene DNA sequences from two or more different AAV serotypes, AAV chimeras, or other AAVs. In some embodiments, a haploid AAV may be composed of DNA-shuffled VP1, DNA-shuffled VP2, and / or DNA-shuffled VP3. In some embodiments, the VP1 protein of the rational polyploid virions disclosed herein is modified, and VP3 is not a modified protein. In some embodiments, the rational polyploid virion comprises a modified VP3 protein and the VP1 is unmodified. In some embodiments, the rational polyploid virion disclosed herein comprises a modified VP1 protein and a modified VP3 protein.

[0267] Examples of modified VP1 proteins include, but are not limited to, peptide insertions into the VP1 protein. Such peptides include, but are not limited to, peptides that are targeting peptides, such as the peptides disclosed herein that target cells of the CNS or PNS. In some embodiments, the targeting peptide is a BBB-penetrating peptide, such as the RVG-9R peptide or a variant thereof, as disclosed in U.S. Patent No. 8,748,567 or U.S. Patent No. 9,757,470, which are incorporated herein by reference in their entireties. In some embodiments, the peptide can be inserted into or substituted at any position selected from amino acid residues 450-480, amino acid residues 575-600 (numbering based on AAV8 VP1 numbering) of the native AAV8 VP1 capsid protein and / or AAV8 VP2 capsid protein of the polyploid virion, or the corresponding positions of a capsid protein from another AAV.

[0268] In another embodiment, nucleic acids encoding VP1, VP2, and / or VP3 can be generated by DNA shuffling. In one embodiment, the first nucleic acid generated by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid generated by DNA shuffling will encode VP2 and VP3. In another embodiment, the first nucleic acid generated by DNA shuffling will encode VP1. In this same embodiment, the second nucleic acid generated by DNA shuffling will encode VP2, and the third nucleic acid will encode VP3. In a further embodiment, the first nucleic acid generated by DNA shuffling will encode VP1 and VP2, and the second nucleic acid generated by DNA shuffling will encode VP3. In an additional embodiment, the first nucleic acid generated by DNA shuffling will encode VP1 and VP3, and the second nucleic acid generated by DNA shuffling will encode VP2.

[0269] In embodiments of the present invention, the rational polyploid vectors disclosed herein exhibit increased transduction of one or more tissues in the CNS and / or peripheral nervous system (PNS). In some embodiments, the rational polyploid vectors disclosed herein exhibit enhanced transduction of one or more of a subject's cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord in vivo after systemic or intrathecal administration compared to non-rational polyploid AAV particles that lack the ability to efficiently cross the blood-brain barrier. In some embodiments, the rational polyploids or polyploid vectors disclosed herein have transduction levels in the brain and / or spinal cord that are at least about 50%, 60%, 70%, 80%, 90%, or 95%, or 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or greater than 1000-fold higher than the parent AAV vector.

[0270] In certain embodiments, modified AAV capsid proteins of the invention comprise one or more modifications of the amino acid sequence of triad loop 4 (between amino acids 575 and 600, inclusive, of the native AAV8 VP1 capsid protein, or the corresponding region of a capsid protein from another AAV). As used herein, a "modification" of an amino acid sequence includes substitutions, insertions, and / or deletions, each of which may involve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In certain embodiments, the modification is a substitution. For example, in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids from triad loop 4 from one AAV may be substituted at any position selected from amino acid residues 450-480, amino acid residues 575-600 (numbering based on AAV8 VP1 numbering) of the native AAV8 VP1 capsid protein and / or AAV8 VP2 capsid protein of a polyploid virion, or the corresponding positions of a capsid protein from another AAV. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids may be inserted at any position selected from amino acid residues 450-480, 575-600, or 575-600 of the native AAV8 VP1 and / or AAV8 VP2 viral structural proteins of a rational polyploid virion. In some embodiments, the insertion is a peptide, including, but not limited to, a targeting peptide or a BBB-penetrating peptide. In some embodiments, the insertion is an RVG-9R peptide, such as those disclosed in U.S. Patent No. 8,748,567 or U.S. Patent No. 9,757,470, which are incorporated herein by reference in their entireties.However, the modified viral capsids of the present invention are not limited to AAV capsids in which amino acids from one AAV capsid have been substituted into another AAV capsid; the substituted and / or inserted amino acids may be from any source and may be naturally occurring or partially or completely synthetic.

[0271] The nucleic acid and amino acid sequences of the capsid proteins from several AAVs described herein are known in the art. Thus, the amino acids "corresponding" to amino acids 575-600 (inclusive) or 585-590 (inclusive) of the native AAV8 capsid protein can be readily determined for any other AAV (e.g., by sequence alignment).

[0272] In some embodiments, the present invention contemplates that the modified capsid proteins of the present invention can be produced by modifying the capsid protein of any AAV currently known or later discovered. Furthermore, the AAV capsid protein to be modified can be, but is not limited to, a naturally occurring AAV capsid protein (e.g., AAV8, AAVrhlO, AAVrh74, AAV3a or 3b, AAV9 capsid protein, or any of the AAVs listed in Table 1). Various manipulations of AAV capsid proteins are known in the art, and those skilled in the art will understand that the present invention is not limited to modifying naturally occurring AAV capsid proteins. For example, the capsid protein to be modified can already be altered compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, e.g., AAV8, AAVrhlO, AAVrh74, AAV3a or 3b, AAV9, or any other AAV currently known or later discovered). Such AAV capsid proteins are also within the scope of the present invention.

[0273] For example, in some embodiments, the AAV capsid protein to be modified may contain an amino acid insertion immediately after amino acid 264 of the native AAV8 capsid protein sequence (see, e.g., PCT Publication WO2006 / 066066) and / or may be an AAV with an altered HI loop as described in PCT Publication WO2009 / 108274, and / or may be an AAV that has been modified to contain a poly-His sequence to facilitate purification. As another illustrative example, an AAV capsid protein may have a peptide targeting sequence incorporated therein as an insertion or substitution. Additionally, an AAV capsid protein may contain a large domain from another AAV substituted and / or inserted into the capsid protein.

[0274] Thus, in certain embodiments, the AAV capsid protein, e.g., the VP3 protein to be modified, may be derived from a naturally occurring AAV, but may further comprise one or more foreign sequences (e.g., foreign to the native virus) inserted into and / or substituted into the capsid protein and / or altered by deletion of one or more amino acids.

[0275] In some embodiments, the rational polyploid virion comprises an AAV rhesus modified or mutant VP3 structural protein, wherein VP1 and VP2 are not AAV rhesus serotypes. In certain embodiments, the mutant VP3 capsid protein is a mutant AAVrh10VP3 or mutant AAVrh74 VP3 viral structural protein. In certain embodiments, the AAV rhesus mutant VP3 comprises at least one mutation in an amino acid corresponding to an amino acid selected from the group consisting of N263, G264, T265, S266, G268, T270, T274, E533, R727, and N737, where all amino acid positions correspond to those of native VP3. In some embodiments, the AAV rhesus mutant VP3 viral structural protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the mutations at amino acid positions corresponding to amino acid positions N263, G264, T265, S266, G268, T270, T274, E533, R727, and N737 of the native VP1 sequence numbering of AAV rh10. In some embodiments, the AAV rhesus rh10 mutant VP3 comprises a mutation selected from the group consisting of N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R727H, and N737P. In exemplary embodiments, the AAV rhesus rh10 mutant VP3 comprises N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R727H, and N737P (AAVrh10LP2, or rh10LP2). In some embodiments, the modified AAVrh10 VP3 structural protein comprises the amino acids of SEQ ID NO: 14 (rh10-LP2 VP3), or of a protein having at least 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 14. In some embodiments, the modified AAVrh10 VP3 structural protein comprises the amino acids of SEQ ID NO: 14 (rh10-LP2 VP3), or of a protein having at least 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 14.

[0276] In some aspects of the embodiments, the AAV rhesus mutant VP3 comprises mutations at amino acids corresponding to amino acids selected from the group consisting of N263, G264, T265, S266, G268, T270, T274, E533, R726, and N736, where all amino acid positions correspond to the native VP1 sequence numbering of AAV rh74. In some embodiments, the AAV rhesus mutant VP3 viral structural protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all of the mutations at amino acid positions corresponding to amino acid positions N263, G264, T265, S266, G268, T270, T274, E533, R726, and N736 of the native VP1 sequence numbering of AAV rh74.

[0277] In some embodiments, the AAV rhesus rh74 mutant VP3 comprises a mutation selected from the group consisting of N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, and N736P. In an exemplary embodiment, the AAV rhesus rh74 mutant VP3 comprises N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, and N736P (AAVrh74LP2, or rh74LP2). Numbering herein is based on rh74 VP1 numbering. In some embodiments, the modified AAVrh74 VP3 structural protein comprises the amino acids of SEQ ID NO: 15 (rh74vv-LP2 VP3), or of a protein having at least 85%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 15. In some aspects of the embodiment, the mutations are located in loop 1 (VRI), or in VR-IV, or in the C-terminal domain at the extreme end of the VP3 viral structural protein. In some aspects of the embodiment, the mutations are located in at least two of these regions.

[0278] In yet another aspect of the embodiment, the mutant AAVrh74VP3 viral structural protein further comprises a mutation in which the W or tryptophan at position 581 is replaced with two subsequent valine (VV) residues. The rh74VP3 mutation consisting of W581VV, N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, and N736P is synonymously referred to as AAVrh74VVLP2 (or rh74VVLP2). In some embodiments, the viral structural protein VP3 of the rational polyploid virions of the invention comprises rh74VVLP2. The present inventors rationally designed polyploid virions containing rh10VP3LP2, rh74VP3LP2, and rh74VVP3LP2 and tested their properties, including their antigenicity, e.g., escape from neutralizing antibodies, and / or humoral immune responses. In some embodiments, rational AAV polyploid virions for use in the methods and compositions disclosed herein are selected from any of AAV8-8-rh10, AAV 8-8-rh74, AAV8-8-rh74vv, AAV 8-8-rh10LP2, AAV 8-8-rh74LP2, AAV 8-8-rh74LP2 vv, and are interchangeably referred to as haploid AAV8-8-rh10, AAV 8-8-rh74, AAV8-8-rh74vv, AAV 8-8-rh10LP2, AAV 8-8-rh74LP2, AAV 8-8-rh74LP2 vv virions.

[0279] In all aspects of the present invention, the VP1, VP2, and VP3 viral structural proteins are used interchangeably with the VP1, VP2, and VP3 capsid proteins. Alternatively, the terms "viral capsid proteins" and "viral structural proteins" are used interchangeably herein to refer to the VP1, VP2, and VP3 viral structural proteins (or structural viral proteins).

[0280] Thus, when a specific AAV capsid protein is referred to herein (e.g., a VP3 protein selected from any of AAV1, AAV6, AAV6.2, AAV7, AAV8, AAV9, rAAVrhlO, rAAVrh74, rAAVrh39, rAAVrh4, or a VP1 or VP2 capsid protein from any of the AAVs shown in Table 1, etc.), it is intended to encompass the native capsid protein as well as capsid proteins with modifications other than those of the present invention. Such modifications include substitutions, insertions, and / or deletions. In certain embodiments, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acids inserted therein relative to the native AAV capsid protein sequence (other than the insertions of the invention). In embodiments of the invention, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60, or less than 70 amino acid substitutions (other than amino acid substitutions according to the invention) compared to the native AAV capsid protein sequence. In embodiments of the invention, the capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more than 20, more than 30, more than 40, more than 50, more than 60, or more than 70 amino acid deletions compared to the native AAV capsid protein sequence (other than the amino acid deletions of the invention).

[0281] Using AAV serotype 8 as an exemplary serotype for VP1 and VP2 in the rational polyploid vectors disclosed herein, M1 is the VP1 start codon, T138 is the VP2 start codon, and M203 and M211 are the VP3 start codons. While deletion of the start codons, typically by substitution of M1 and T138, renders VP1 and VP2 expression inoperative, similar deletion of the VP3 start codon is not sufficient. This is because the viral capsid ORF contains numerous ATG codons with varying strengths as start codons. Therefore, when designing constructs that do not express VP3, care must be taken to ensure that alternative VP3 species are not produced. For VP3 where loss of either M204 or M212 is necessary, or if VP2 but not VP3 is desired, deleting M204 and M212 is generally the best approach (Warrington, KH Jr., et al., J. of Virol. 78(12): 6595-6609 (June 2004)). This can be done by mutation, e.g., substitution, or other means known in the art. Corresponding start codons in other serotypes can be readily determined, as can whether additional ATG sequences, such as those in VP3, can serve as alternative start codons.

[0282] Thus, for example, the term "AAV8 capsid protein" includes AAV capsid proteins having the native AAV8 capsid protein sequence (see native AAV8 VP1 capsid protein: GenBank Accession No. AF513852.1, Protein ID: AAN03856.1), as well as those that contain substitutions, insertions, and / or deletions (as described in the paragraph above) in the native AAV8 capsid protein sequence.

[0283] In certain embodiments, the AAV capsid protein has a native AAV capsid protein sequence or has an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% similar or identical to a native AAV capsid protein sequence. For example, in certain embodiments, "AAV8" capsid protein encompasses native AAV8 capsid protein sequences as well as sequences that are at least about 75%, 80%<85%, 90%, 95%, 97%, 98%, or 99% similar or identical to a native AAV8 capsid protein sequence.

[0284] Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2,482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), by the BestFit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection.

[0285] Another suitable algorithm is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are set to default values ​​as necessary. The parameters are dynamic values ​​established by the program itself depending on the composition of the specific sequence and the configuration of the specific database in which the sequence of interest is searched, but the values ​​can be adjusted to increase sensitivity.

[0286] Furthermore, an additional useful algorithm is Gapped BLAST as reported by Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402.

[0287] The present invention also provides viral capsids comprising, consisting essentially of, or consisting of modified AAV capsid proteins of the present invention. In certain embodiments, the viral capsid is a parvovirus capsid, which may further be an autonomous parvovirus capsid or a dependent virus capsid. Optionally, the viral capsid is an AAV capsid. In certain embodiments, the AAV capsid comprises a VP3 protein selected from AAV1, AAV6, AAV6.2, AAV7, AAV9, rAAVrhlO, rAAVrh74, rAAVrh39, or rAAVrh43, and VP1 and / or VP2 from any other AAV listed in Table 1 or otherwise known or later discovered, and / or is derived from any of the foregoing by one or more insertions, substitutions, and / or deletions.

[0288] In embodiments of the invention, the isolated AAV virions, or substantially homogeneous populations of AAV virions, are not the product of expression of a mixture of one nucleic acid helper plasmid expressing VP1, VP2, and VP3 of one serotype and another nucleic acid helper plasmid expressing VP1, VP2, and VP3 of another serotype; such expression is referred to as "cross-dressing."

[0289] In embodiments of the invention, the isolated AAV virions do not comprise mosaic capsids, and the substantially homogeneous population of AAV virions does not comprise a substantially homogeneous population of mosaic capsids. VI. Rational Polyploid AAV Vectors for Treating CNS Disorders and Diseases

[0290] In some embodiments, a rational polyploid vector may comprise a capsid that includes (e.g., substituted or inserted into) a targeting sequence (also called a targeting peptide) that directs the viral capsid to interact with a cell surface molecule present on a desired target tissue (see, e.g., International Patent Publication No. WO 00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774; Shi et al., Human Gene Therapy 17:353-361 (2006) [describing insertion of the integrin receptor binding motif RGD at positions 520 and / or 584 of the AAV capsid subunit]; and U.S. Patent No. 7,314,912 [describing insertion of a P1 peptide containing an RGD motif after amino acids 447, 534, 573, and 587 of the AAV2 capsid subunit]). Other positions within the AAV capsid subunit that tolerate insertions are known in the art (eg, positions 449 and 588 described by Grifman et al., Molecular Therapy 3:964-975 (2001)).

[0291] For example, some viral capsids of the present invention have relatively inefficient tropism for most target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lung). Advantageously, targeting sequences can be incorporated into these low-transduction vectors to confer the desired tropism, and, if necessary, selective tropism for specific tissues, to the viral capsid. AAV capsid proteins, capsids, and vectors containing targeting sequences are described, for example, in International Patent Publication WO 00 / 28004. Another possibility is to incorporate one or more non-naturally occurring amino acids, such as those described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006), into the orthogonal site of an AAV capsid subunit as a means of redirecting the low-transduction vector to the desired target tissue. Advantageously, these unnatural amino acids can be used to chemically link molecules of interest to AAV capsid proteins, including, but not limited to, glycans (mannose - dendritic cell targeting); RGD, bombesin, or neuropeptides for targeted delivery to specific cancer cell types; RNA aptamers or peptides selected from phage display targeted to specific cell surface receptors, e.g., growth factor receptors, integrins, etc. Methods for chemically modifying amino acids are known in the art (see, e.g., Greg T. Hermanson, Bioconjugate Techniques, 1999). st edition, Academic Press, 1996).

[0292] In representative embodiments, the targeting sequence can be a viral capsid sequence (e.g., an autonomous parvovirus capsid sequence, an AAV capsid sequence, or any other viral capsid sequence) that directs infection to a specific cell type.

[0293] B19 uses globoside as its receptor to infect primary erythroid progenitor cells (Brown et al., (1993) Science 262:114). The structure of B19 has been determined to 8 Å resolution (Agbandje-McKenna et al., (1994) Virology 203:106). The region of the B19 capsid that binds globoside has been mapped to amino acids 399–406 (Chipman et al., (1993) Virology 194:419), which is the loop-out region between beta-barrel structures E and F (Chipman et al., (1996) Proc. Nat. Acad. Sci. USA 93:7502). Thus, the globoside receptor binding domain of the B19 capsid can be substituted into an AAV capsid protein to target viral capsids or viral vectors containing it to erythroid cells.

[0294] In representative embodiments, the exogenous targeting sequence may be any amino acid sequence encoding a peptide that alters the tropism of a viral capsid or viral vector, including a modified AAV capsid protein. In certain embodiments, the targeting peptide or protein may be naturally occurring or, alternatively, may be wholly or partially synthetic. Exemplary targeting sequences include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as RGD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor I and II, etc.), cytokines, melanocyte-stimulating hormones (e.g., α, β, or γ), neuropeptides, and endorphins, as well as fragments thereof that retain the ability to target cells to their cognate receptors. Other illustrative peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin-releasing peptide, interleukin 2, hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin, β-endorphin, leucine enkephalin, limorphin, α-neo-enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof described above. Additionally, as a further alternative, a binding domain from a toxin (e.g., tetanus toxin or snake venom, such as α-bungarotoxin) can be substituted into the capsid protein as a targeting sequence. In still further exemplary embodiments, the AAV capsid protein may be modified by the substitution of a "non-classical" import / export signal peptide (e.g., fibroblast growth factor-1 and -2, interleukin-1, HIV-1 Tat protein, herpesvirus VP22 protein, etc.) into the AAV capsid protein, as described by Cleves (Current Biology 7:R318 (1997)). Peptide motifs that direct uptake by specific cells are also encompassed; for example, the FVFLP peptide motif (SEQ ID NO: 26) induces uptake by liver cells.

[0295] Phage display techniques, as well as other techniques known in the art, can be used to identify peptides that recognize any cell type of interest.

[0296] The targeting sequence can encode any peptide that targets a cell surface binding site, including a receptor (e.g., a protein, carbohydrate, glycoprotein, or proteoglycan). Examples of cell surface binding sites include, but are not limited to, heparan sulfate, chondroitin sulfate, and other glycosaminoglycans; sialic acid moieties found on mucins, glycoproteins, and gangliosides; MHC I glycoproteins; carbohydrate moieties found on membrane glycoproteins, including mannose, N-acetyl-galactosamine, N-acetylglucosamine, fucose, galactose, and the like.

[0297] In certain embodiments, a heparan sulfate (HS) or heparin-binding domain is substituted into the viral capsid (e.g., into an AAV that does not otherwise bind HS or heparin). It is known in the art that HS / heparin binding is mediated by a "basic patch" rich in arginine and / or lysine. In an exemplary embodiment, the sequence follows the motif BXXB, where "B" is a basic residue and X is neutral and / or hydrophobic. As a non-limiting example, BXXB is RGNR (SEQ ID NO: 27). In certain embodiments, BXXB is substituted at amino acid positions 262 to 265 in the native AAV2 capsid protein, or at the corresponding position in the capsid protein of another AAV.

[0298] Furthermore, as a further alternative, the targeting sequence may be a peptide (e.g., which may contain arginine and / or lysine residues that can be chemically coupled via their R groups) that can be used for chemical coupling to another molecule for cell entry.

[0299] Alternatively, the AAV capsid proteins or viral capsids of the invention can contain mutations such as those described in WO2006 / 066066. For example, the capsid protein can contain selective amino acid substitutions at amino acid positions 263, 705, 708, and / or 716 of the native AAV2 capsid protein, or corresponding changes in a capsid protein from another AAV. Additionally or alternatively, in representative embodiments, the capsid protein, viral capsid, or vector contains a selective amino acid insertion immediately after amino acid position 264 of the AAV2 capsid protein, or a corresponding change in a capsid protein from another AAV. By "immediately after amino acid position X," it is intended that the insertion is immediately after the indicated amino acid position (e.g., "after amino acid position 264" indicates a point insertion at position 265, or a larger insertion, such as positions 265-268). The above-described embodiments of the invention can be used to deliver heterologous nucleic acids to cells or subjects as described herein.

[0300] In some embodiments, the rational polyploid or haploid vectors disclosed herein can be used to deliver heterologous nucleic acids to cells, including neural or non-neuronal cells, in the CNS and / or peripheral nervous system. In some embodiments, the rational polyploid or haploid vectors disclosed herein are useful for treating medical conditions or diseases associated with abnormal gene expression in CNS tissues or cells and / or PNS tissues or cells. CNS cells can be, for example, neurons, astrocytes, paradendrocytes, ependymal cells, or microglial cells. CNS tissues can be, for example, more than the cortex, striatum, thalamus, medulla, hippocampus, midbrain, Purkinje tissue, cerebellum, and spinal cord of a subject, including the cervical, thoracic, and lumbar spinal cord of the CNS, as well as the medulla and choroid plexus.

[0301] For example, the rational polyploid or haploid vectors disclosed herein can be used to treat brain diseases, including brain cancers and brain cancers (e.g., glioblastoma), neurodegenerative diseases, including, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, amytrophic lateral sclerosis (ALS), and dopamine transporter deficiency syndrome (a form of childhood parkinsonism caused by loss-of-function mutations in a single gene, DAT1 / SLC6A3). Other medical conditions or diseases of the CNS useful for treatment using the rational polyploid or haploid vectors disclosed herein include, for example, neurological diseases and / or disorders. Such neurodegenerative diseases and / or disorders include, but are not limited to, dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathies, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff disease, Charcot-Marie-Tooth disease, Angelman syndrome, Canavan disease, late-onset childhood neuronal ceroid lipofuscinosis, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodystrophy, hereditary lysosomal storage diseases, e.g., Niemann-Pick disease type C1, and / or neuronal ceroid lipofuscinoses, e.g., Batten disease, progressive supranuclear palsy, corticobasal syndrome, and brain cancers (including astrocytoma and glioblastoma). In some preferred embodiments of the invention, the gene encodes a therapeutic expression product, preferably a therapeutic polypeptide suitable for use in the treatment of diseases or conditions associated with aberrant gene expression, optionally in the CNS.

[0302] In some embodiments, the rational polyploid or haploid vectors disclosed herein can be used to express therapeutic expression products useful for treating CNS diseases. The term "CNS disease" is understood in principle by those skilled in the art. This term relates to diseases that can be treated and / or prevented by administering an active compound to the CNS, particularly to CNS cells. In some embodiments, the CNS disease is a neurodegenerative disease and / or disorder.

[0303] By way of non-limiting example, the CNS disease may be selected from the following: septum pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupil, Addie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Gouttiere syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis ALS, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or cerebellar-spinal degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic neuropathy, back pain, Barth syndrome, Batten disease, Becker's myotonia, Behcet's disease Disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernard-Ross syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, intrapartum brachial plexus injury, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysms, brain injury, Brown-Séquard syndrome, spinal-bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous vascular malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, craniopathy, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysms aneurysms), cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformations, cerebral gigantism, hypoxic encephalopathy, cerebral palsy, cerebro-oculofacial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disease, chorea, chorea acanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic dysregulation, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, agenesis of the corpus callosum, coma, complex regional pain syndrome, congenital facial diplegia, congenital myasthenia gravis, congenital myopathy, congenital cavernous malformations,Corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalic inclusion disease, cytomegalovirus infection, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, dementia, dementia-multiinfarct, dementia-semantic, dementia-subcortical, dementia with Lewy bodies, dentate cerebellar ataxia disease, dentatorubral atrophy, dermatomyositis, developmental apraxia, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, autonomic neuropathy, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalopathy (familial infantile), trigeminal angiomatosis, epilepsy, Epileptic hemiplegia, Erb's palsy, Erb-Dusenne and Degerines-Klumpke palsy, essential tremor, extrapontine myelinolysis, Fabry's disease, Fahr's syndrome, syncope, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile seizures, fibromuscular dysplasia, Fisher's syndrome, limp infant syndrome, foot drop, Friedreich's ataxia, frontotemporal dementia, Gaucher's disease, generalized Gangliosidosis, Gerstmann syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemicrania, hemifacial spasm, alternating hemiplegia, hereditary neuropathies, hereditary spastic paraplegia, hereditary polyneuropathy ataxia, herpes zoster Zoster), herpes zoster oticus, Hirayama syndrome, Holmes-Addie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydroanencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelopathy, hyperadrenocorticism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease, infantile spasms, inflammatory myopathy, occipital forencephalon prolapse,Intestinal lipodystrophy, Intracranial cyst, Intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, Klein-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, Kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral femoral cutaneous nerve entrapment, Lateral cord syndrome, Learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Levine-Critchley syndrome, Dementia with Lewy bodies, Lipid storage disease, Lipoproteinosis, Lissencephaly, Locked-in syndrome, Lou Gehrig's disease, Lupus Neurological sequelae, Lyme disease - neurological complications, Machado-Joseph disease, megalencephaly, megalencephaly, Melkerson-Rosenthal syndrome, meningitis, meningitis and encephalitis, Menkes disease, dyssensory femoral neuralgia, metachromatic leukodystrophy, microcephaly, migraine, Miller-Fisher syndrome, minor stroke, mitochondrial myopathy, Moebius syndrome, unilateral muscular atrophy, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuron disease Pathopathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, muscular dystrophy - congenital, gravitational myasthenia, diffuse myeloablative sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myopathy - congenital, myopathy - thyrotoxicosis, myotonia, myotonia congenital, narcolepsy, neuroacanthocytosis, neurodegeneration with cerebral iron deposition, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease Complications, Neurological consequences of cytomegalovirus infection, Neurological manifestations of Pompe disease, Neurological sequelae of lupus, Neuromyelitis optica, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorders, Neuropathies - hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, Occipital neuralgia, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsoclonus-myoclonus, Orthostatic hypotension, Overuse syndrome, Pain - chronic,Pantothenate kinase-related neurodegeneration, paraneoplastic syndromes, abnormal sensations, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg disease, Pelizaeus-Merzbacher disease, Pena-Shocker II syndrome, nerve root cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, compressed nerve, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, sensitization Postinfectious encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural orthostatic tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive facial hemiatrophy, progressive gait ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudo-Torch syndrome, pseudo-toxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay Hunt syndrome I, Ramsay Hunt syndrome II, Rasmussen encephalitis, reflex sympathetic dystrophy Trophy syndrome, Refsum's disease, Refsum's disease-infant, repetitive movement disorder, repetitive stress disorder, restless legs syndrome, retroviral-associated myelopathy, Rett's syndrome, Reye's syndrome, rheumatoid encephalitis, Riley-Day syndrome, sacral radicular cyst, St. Vitus chorea, salivary gland disease, Sandhoff's disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy of the infant (SMEI), shaken baby syndrome, shingles (Shin gles), Shy-Drager syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal tumor, spinal muscular atrophy, spinocerebellar atrophy, spinocerebellar degeneration, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short-term, unilateral, neuralgiform (SUNCT) headache, dysphagia Disorder), Sydenham chorea, syncope, syphilitic spinal sclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, spinal fistula, tardive dyskinesia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's myotonia, thoracic outlet syndrome, thyrotoxic myopathy, painful tics, Todd's palsy, Tourette's syndrome,Transient ischemic attacks, transmissible spongiform encephalopathies, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer's syndrome, tuberous sclerosis, vascular erectile tumors, vasculitic syndromes of the central and peripheral nervous system, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsaff syndrome, West syndrome, whiplash injury, Whipple's disease, Williams syndrome, Wilson's disease, Wolman disease, X-linked spinal and bulbar muscular atrophy.

[0304] In some embodiments, the CNS disease is selected from the list consisting of dopamine transporter deficiency syndrome, attention deficit / hyperactivity disorder (ADHD), bipolar disorder, epilepsy, multiple sclerosis, tauopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Krabbe disease, adrenoleukodystrophy, motor neuron disease, cerebral palsy, Batten disease, Gaucher disease, Tay-Sachs disease, Rett syndrome, Sandhoff disease, Charcot-Marie-Tooth disease, Angelman syndrome, Canavan disease, late-onset childhood neuronal ceroid lipofuscinosis, mucopolysaccharidosis IIIA, mucopolysaccharidosis IIIB, metachromatic leukodystrophy, hereditary lysosomal storage diseases such as Niemann-Pick disease type C1, and / or neuronal ceroid lipofuscinosis such as Batten disease, progressive supranuclear palsy, corticobasal syndrome, and brain cancer (including astrocytoma and glioblastoma).

[0305] In some embodiments, the rational polyploid or haploid vectors disclosed herein may be used to express a therapeutic expression product useful in the treatment of a disease selected from methylmalonic acidemia (MMA), alpha 1 antitrypsin deficiency (AATD), or autosomal dominant polycystic kidney disease (ADPKD).

[0306] In some embodiments, the rational polyploid or haploid vectors disclosed herein may be used to express a therapeutic expression product useful in the treatment of a disease selected from any of the muscular dystrophies, including myotonic dystrophy (DM1 and DM2), limb-girdle dystrophy, Dusenne dystrophy, Becker dystrophy, congenital dystrophy, facioscapulohumeral dystrophy, Emilie-Dreifuss dystrophy, distal dystrophy, oculopharyngeal dystrophy, and collagen VI-associated dystrophy.

[0307] In some embodiments, the rational polyploid or haploid vectors disclosed herein may be used to express a therapeutic expression product useful in the treatment of a disease of gastrointestinal origin or a gastrointestinal disorder, for example, any gastrointestinal or small intestinal disorder selected from inflammatory bowel disease (including IBD, ulcerative colitis, and Crohn's disease), irritable bowel syndrome (IBS), celiac disease, hereditary hemochromatosis, Lynch syndrome, familial adenomatous polyposis, juvenile polyposis syndrome, Peutz-Jeghers syndrome, eosinophilic gastrointestinal diseases (e.g., eosinophilic gastroenteritis (EGE)), microvillus inclusion body disease, megacystic bladder disease, microcolon-intestinal hypoperistalsis, mitochondrial neurogastrointestinal encephalopathy syndrome, intestinal lymphangiectasia, autoimmune gastrointestinal motility disorders, tropical sprue, Whipple's disease, lactose intolerance, and hereditary amyloidosis.

[0308] In certain embodiments, the rational polyploid AAV vectors disclosed herein are administered prophylactically to a subject to prevent the onset of a disease. In another embodiment, the AAV particles of the present disclosure are administered to treat (alleviate the effects of) a disease or its symptoms. In yet another embodiment, the rational polyploid AAV vectors disclosed herein are administered to cure (eliminate) a disease. In another embodiment, the rational polyploid AAV vectors disclosed herein are administered to prevent or slow the progression of a disease. In yet another embodiment, the AAV particles of the present disclosure are used to ameliorate the deleterious effects of a disease. Disease status and / or progression can be determined or monitored by standard methods known in the art.

[0309] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation or amelioration of neurological diseases and / or disorders.

[0310] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of tauopathies.

[0311] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of Alzheimer's disease.

[0312] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of Friedreich's ataxia or any disease resulting from the loss or partial loss of frataxin protein.

[0313] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of Parkinson's disease.

[0314] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of amyotrophic lateral sclerosis.

[0315] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of Huntington's disease.

[0316] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation or amelioration of chronic or neuropathic pain.

[0317] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of diseases associated with the central nervous system (CNS).

[0318] In some embodiments, the rational polyploid AAV vectors disclosed herein are useful in the medical field for the treatment, prevention, alleviation, or amelioration of diseases associated with the peripheral nervous system (PNS).

[0319] In certain embodiments, the AAV particles of the present disclosure are administered to a subject having at least one of the diseases or conditions described herein.

[0320] As used herein, any disease associated with the central or peripheral nervous system and its components (eg, neurons) may be considered a "neurological disease."

[0321] Any neurological disease may be treated with the AAV particles of the present disclosure or pharmaceutical compositions thereof, including, but not limited to, septum pellucidum defect, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie pupil, Adie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, antiphospholipid syndrome, aphasia, Apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia, ataxia-telangiectasia, ataxia and cerebellar or cerebellar-spinal degeneration, atrial fibrillation and stroke, attention-deficit hyperactivity disorder, autism spectrum disorder, autonomic neuropathy, back pain, Barth syndrome, Batten disease, Becker's myotonia, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernard-Ross syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury during birth, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysms Aneurysm, brain injury, Brown-Séquard syndrome, bulbar palsy, spinal and bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous vascular malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, craniopathy, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm aneurysms), cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, hypoxic encephalopathy, cerebral palsy, cerebro-oculofacial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Chiari malformation, cholesterol ester storage disease, chorea, chorea acanthocytosis, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic dysregulation, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, agenesis of the corpus callosum, coma, complex regional pain syndrome, concentric sclerosis (barosclerosis), congenital diplegia of the face,Congenital myasthenia, congenital myopathy, congenital vascular cavernous malformation, corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, chronic progressive external ophthalmoplegia, cumulative trauma disorder, Cushing's syndrome, cytomegalic inclusion disease, cytomegalovirus infection, dancing eyes-dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, dementia, dementia-multiple infarct, dementia-semantic, dementia-subcortical, Lewy syndrome Dementia with bodies, demyelinating disease, dentatocerebellar ataxia, dentatorubral atrophy, dermatomyositis, developmental apraxia, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, distal hereditary motor neuron disease, Dravet syndrome, autonomic neuropathy, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalocele, encephalomyelitis, encephalopathy, encephalopathy (familial) Infantile), trigeminal angiomatosis, epilepsy, epileptic hemiplegia, paroxysmal ataxia, Erb's palsy, Erb-Dusenne and Degerines-Klumpke palsy, essential tremor, extrapontine myelinolysis, Farber's disease, Fabry's disease, Fahr's syndrome, syncope, familial dysautonomia, familial hemangiomas, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile convulsions, fibromuscular dysplasia, Fisher's syndrome, limp infant syndrome, foot drop, Friedreich's ataxia, frontotemporal type Dementia, Gaucher disease, systemic gangliosidosis (GM1, GM2), Gerstmann syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemicrania, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, polyneuropathy-type hereditary ataxia, Herpes zoster, Herpes zoster oticus, Hirayama syndrome, Holmes-Addie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, Hurler syndrome, hydroanencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelia, hyperadrenocorticism, hypersomnia, hypertonia,Hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease, infantile spasms, inflammatory myopathy, foramen occipitalis prolapse, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy's disease, Kinsbone syndrome, Klein-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugel syndrome Leberg-Welander disease, Kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral femoral cutaneous nerve entrapment, Lateral cord syndrome, Learning disability, Leigh's disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, Leukodystrophy, Levine-Critchley syndrome, Dementia with Lewy bodies, Lichtheim's disease, Lipid storage disease, Lipoproteinosis, Lissencephaly, Locked-in syndrome, Lou Gehrig's disease, Lupus - neurological sequelae, Lyme disease - neurological complications, Lysosomal storage disorders, Machado-Joseph disease, Megalencephaly, Melkersson-Rosenberg syndrome Mental syndrome, meningitis, meningitis and encephalitis, Menkes disease, dyssensory femoral neuralgia, metachromatic leukodystrophy, microcephaly, migraine, Miller-Fisher syndrome, minor stroke, mitochondrial myopathy, mitochondrial DNA depletion syndrome, Moebius syndrome, unilateral muscular atrophy, Morvan syndrome, motor neuron disease, moyamoya disease, mucolipidosis, mucopolysaccharidoses, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, muscular dystrophy-congenital, myasthenia gravis, diffuse muscular atrophy Cancerous myeloablative sclerosis, myelitis, myoclonic encephalopathy of infancy, myoclonus, myoclonic epilepsy, myopathy, myopathy-congenital, myopathy-thyrotoxic, myotonia, myotonia congenita, narcolepsy, NARP (neuropathy, ataxia and retinitis pigmentosa), neuroacanthocytosis, neurodegeneration with cerebral iron deposition, neurodegenerative diseases, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica,Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorder, Neuropathic pain, Neuropathic - hereditary, Neuropathy, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, Occipital neuralgia, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsoclonus-myoclonus, Orthostatic hypotension, Overuse syndrome, Pain - chronic, Pantothenate kinase-related neurodegeneration, Paraneoplastic syndromes, Dysesthesias, Parkinson's disease, Paroxysmal choreoathetosis, Paroxysmal hemicrania, Parry-Romberg syndrome, Pelizaeus-Meyer syndrome Lutzbacher disease, Pena-Shocker II syndrome, nerve root cyst, peroneal muscular atrophy, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phytanic acid storage disease, Pick's disease, compressed nerve, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, post-infectious encephalomyelitis, postural hypotension, postural orthostatic tachycardia syndrome, postural tachycardia syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive bulbar palsy, progressive facial hemimyopathy, progressive gait ataxia, progressive multifocal leukomalacia Interstitial encephalopathy, Progressive muscular atrophy, Progressive sclerosing poliodystrophy, Progressive supranuclear palsy, Prosopagnosia, Pseudobulbar palsy, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor cerebri, Psychogenic movement syndrome, Ramsay-Hunt syndrome I, Ramsay-Hunt syndrome II, Rasmussen encephalitis, Reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease-infantile, Repetitive movement disorder, Repetitive stress disorder, Restless legs syndrome, Retroviral-associated myelopathy, Rett syndrome, Reye syndrome, Rheumatic encephalitis, Riley-Day syndrome, Sacral radicular cyst, Saint Vitus chorea, Salivary gland disease, Sandhoff disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy in infants (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal tumor, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, isolated ataxia, Steele-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke,Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short-term, unilateral, neuralgiform (SUNCT) headache, swallowing disorder, Sydenham chorea, syncope, syphilitic spinal sclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, fistula, tardive dyskinesia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's myotonia, thoracic outlet syndrome, thyrotoxic myopathy, painful tics, Todd's palsy, Tourette's syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic ataxia Total paraplegia, Troyer's syndrome, tuberous sclerosis, vascular erectile tumors, vasculitic syndromes of the central and peripheral nervous system, vitamin B12 deficiency, von Economo disease, von Hippel-Lindau disease (VHL), von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsaff syndrome, West syndrome, whiplash injury, Whipple's disease, Williams syndrome, Wilson's disease, Wolman disease, X-linked spinal and bulbar muscular atrophy.

[0322] In some embodiments, the therapeutic gene or expression product encoded by the rational polyploid or haploid vectors disclosed herein may be selected from the group consisting of NPC1, EAAT2, NPY, CYP46A1, GLB1, APOE (e.g., ApoE2, ApoE3, or ApoE4), HEX, CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, SUMF1, DCTN1, PRPH, SOD1, NEFH, GBA, IDUA, NAGLU, GUSB, ARSA, MANB, AADC, GDNF, SOD1, NTN, ASP, MAPT, APOE, HTT, MECP2, PTCHD1, GJB1, UBE3A, HEXA, MOG.

[0323] Additionally or alternatively, the expression product may be an antibody, antibody fragment, or antibody-like scaffold protein. In some embodiments, exemplary polypeptide expression products include neuroprotective polypeptides and anti-angiogenic polypeptides. In one aspect, the rational polyploid vectors disclosed herein comprise a viral genome encoding a polypeptide payload. The polypeptide may be, but is not limited to, an antibody, aromatic L-amino acid decarboxylase (AADC), survival motor neuron 1 (SMN1), frataxin (FXN), ApoE2, GBA1, GRN, ASP A, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, or GAN.

[0324] Additional suitable polypeptides include, but are not limited to, glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), neurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelium-derived factor (PEDF), Wnt polypeptides, soluble Fit-1, angiostatin, endostatin, VEGF, anti-VEGF antibodies, soluble VEGFR, Factor VIII (FVIII), Factor IX (FIX), and members of the hedgehog family (such as sonic hedgehog, Indian hedgehog, and desert hedgehog).

[0325] In some embodiments, useful therapeutic expression products include hormones and growth and differentiation factors, including, but not limited to, insulin; glucagon; growth hormone (GH); parathyroid hormone (PTH); growth hormone-releasing factor (GRF); follicle-stimulating hormone (FSH); luteinizing hormone (LH); human chorionic gonadotropin (hCG); vascular endothelial growth factor (VEGF); angiopoietin; angiostatin; granulocyte colony-stimulating factor (GCSF); erythropoietin (EPO); connective tissue growth factor (CTGF); basic fibroblast growth factor (bFGF); acidic fibroblast growth factor (aFGF); epidermal growth factor (EGF); platelet-derived growth factor (PDGF); insulin growth factor I and II (IGF-I and IGF-II); TGFa These include any one of the transforming growth factor alpha superfamily, including activin; inhibin; or any of the bone morphogenetic proteins (BMPs) BMP1-15; any one of the heregulin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors; nerve growth factor (NGF); brain-derived neurotrophic factor (BDNF); neurotrophins NT-3 and NT-4 / 5; chorionic neurotrophic factor (CNTF); glial cell line-derived neurotrophic factor (GDNF); neurturin; agrin; any one of the semaphorin / collapsin family; netrin-1 and netrin-2; hepatocyte growth factor (HGF); ephrin; noggin; sonic hedgehog; and tyrosine hydroxylase.

[0326] Additionally or alternatively, the therapeutic gene or expression product encoded by the rational polyploid or haploid vector disclosed herein can be a gene editing system (e.g., CRISPR-Cas9 system, TALEN, ZFN, etc.) directed to a disease allele. Additionally or alternatively, the expression product can be one or more regulatory polynucleotides, such as RNA or DNA molecules as therapeutic agents. For example, the regulatory polynucleotide can be miRNA or siRNA. The target gene can be any gene associated with any neurological disease, including, but not limited to, those listed herein. For example, siRNA duplexes or encoded dsRNA can reduce or silence target gene expression in CNS cells, thereby improving the symptoms of the neurological disease. In one non-limiting example, the target gene is huntingtin (HTT). In another non-limiting example, the target gene is microtubule-associated protein tau (MAPT). In some embodiments, useful expression products include proteins that regulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12, and IL-18), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factor alpha and beta, interferons (alpha, beta, and gamma), stem cell factor, and flk-2 / flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T-cell receptors, chimeric T-cell receptors, single-chain T-cell receptors, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins, such as complement regulatory protein, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1, CF2, and CD59.

[0327] In some embodiments, useful expression products include any one of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and / or lipid modulation, including low-density lipoprotein (LDL) receptor, high-density lipoprotein (HDL) receptor, very-low-density lipoprotein (VLDL) receptor, and scavenger receptors. The present invention also encompasses the use of gene products such as members of the steroid hormone receptor superfamily, including glucocorticoid receptor and estrogen receptor, vitamin D receptor, and other nuclear receptors. Additionally, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP-2, myb, MyoD, and myogenin, ETS box-containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT box-containing proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS-binding proteins, STATs, GATA box-binding proteins such as GATA-3, and the forkhead family of winged helix proteins.

[0328] In some embodiments, useful expression products include non-naturally occurring polypeptides, such as chimeric or hybrid polypeptides having non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions. Further suitable expression products include microRNAs (miRNAs), interfering RNAs, antisense RNAs, ribozymes, and aptamers.

[0329] In alternative embodiments, the rational polyploid or haploid vectors disclosed herein can be used to deliver heterologous nucleic acids to cells in a subject in vivo. In some embodiments, the rational polyploid or haploid vectors disclosed herein can be used to treat any one or more of the following diseases as described herein: lysosomal storage disorders, e.g., mucopolysaccharidosis disorders (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [α-L-iduronidase], Scheie syndrome [α-L-iduronidase], Hurler-Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilippo syndrome A [heparin sulfatase], amidase], B [N-acetylglucosaminidase], C [acetyl-CoA; α-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [β-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (e.g., Pompe disease; lysosomal acid α-glucosidase).

[0330] Those skilled in the art will understand that for some AAV capsid proteins, the corresponding modifications will be insertions and / or substitutions, depending on whether the corresponding amino acid positions are partially or completely present in the virus, or alternatively, completely absent. Similarly, when modifying an AAV other than AAV2, a particular amino acid position may differ from its position in AAV2 (see, for example, Table 3). As discussed elsewhere herein, the corresponding amino acid positions will be readily apparent to those skilled in the art using well-known techniques.

[0331] In representative embodiments, the insertion and / or substitution and / or deletion in the capsid protein results in the insertion, substitution and / or rearrangement of (i) amino acids that maintain the hydrophilic loop structure within that region; (ii) amino acids that change the configuration of the loop structure; (iii) charged amino acids; and / or (iv) amino acids that can be phosphorylated or sulfated, or otherwise changed by post-translational modification (e.g., glycosylation) after 264 in the AAV2 capsid protein or a corresponding change in the capsid protein of another AAV. Amino acids suitable for insertion / substitution include aspartic acid, glutamic acid, valine, leucine, lysine, arginine, threonine, serine, tyrosine, glycine, alanine, proline, asparagine, phenylalanine, tyrosine, or glutamine. In certain embodiments, threonine is inserted or substituted into the capsid subunit. Non-limiting examples of corresponding positions in several other AAVs are shown in Table 3 (position 2). In specific embodiments, the amino acid insertion or substitution is threonine, aspartic acid, glutamic acid, or phenylalanine (excluding AAV, which has a threonine, glutamic acid, or phenylalanine at this position).

[0332] In a further embodiment, the modified capsid protein or capsid may comprise mutations as described in WO2009 / 108274.

[0333] Alternatively, the AAV capsid protein may contain a mutation such as those described by Zhong et al. (Virology 381: 194-202 (2008); Proc. Nat. Acad. Sci. 105: 7827-32 (2008)). For example, the AAV capsid protein may contain a YF mutation at amino acid position 730.

[0334] The above modifications can be incorporated into the capsid proteins or capsids of the present invention in combination with each other and / or in combination with any other modification now known or later discovered.

[0335] The present invention also encompasses viral vectors comprising the modified capsid proteins and capsids of the present invention. In certain embodiments, the viral vector is a parvovirus vector (e.g., comprising a parvovirus capsid and / or vector genome), for example, an AAV vector (e.g., comprising an AAV capsid and / or vector genome). In a representative embodiment, the viral vector comprises a modified AAV capsid comprising the modified capsid protein subunits of the present invention and a vector genome.

[0336] For example, in a representative embodiment, the viral vector comprises (a) a modified viral capsid (e.g., a modified AAV capsid) comprising a modified capsid protein of the invention, and (b) a nucleic acid comprising a terminal repeat sequence (e.g., an AAV TR), wherein the nucleic acid comprising the terminal repeat sequence is encapsidated by the modified viral capsid. The nucleic acid may optionally comprise two terminal repeats (e.g., two AAV TRs).

[0337] In representative embodiments, the viral vector is a recombinant viral vector that contains a heterologous nucleic acid encoding a polypeptide or functional RNA of interest. Recombinant viral vectors are described in more detail below.

[0338] In some embodiments, the rational polyploid (e.g., rational haploid) AAV vectors disclosed herein have: (i) an increased ability to cross the BBB after systemic or intrathecal administration compared to a non-polyploid parental AAV vector; (ii) increased biodistribution in the CNS or PNS or both, and / or an increased ability to transduce brain tissue (e.g., neurons and non-neuronal cells) compared to the level of transduction by a non-haploid parental AAV vector; (iii) enhanced systemic transduction by the viral vector compared to the level of transduction observed by a non-rational polyploid parental AAV vector in an animal subject; (iv) a reduced humoral immune response compared to the level of immune response elicited by a non-rational polyploid parental AAV vector; and (v) reduced neutralization by a neutralizing antibody against the parental AAV vector compared to the level of neutralization of the non-polyploid parental AAV vector by the same neutralizing antibody.

[0339] Furthermore, in some embodiments of the present invention, the rational polyploid or haploid AAV vectors disclosed herein demonstrate efficient transduction of CNS and / or PNS target tissues, including, but not limited to, the cortex, striatum, thalamus, medulla, hippocampus, midbrain, Purkinje tissue, cerebellum and spinal cord of a subject, including the cervical, thoracic and lumbar spinal cord as well as the medulla and choroid plexus of the CNS.

[0340] In some embodiments, the population of rational polyploid AAV virions allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from the AAV8 viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV rh10 serotype; the population of rational polyploid AAV 8-8-rh10 virions elicits a reduced humoral response compared to the humoral response elicited by the parental AAV8 serotype, and the repeated dosing comprises a first administration of the population of rational polyploid AAV 8-8-rh10 virions and a second administration of parental AAV serotype 8 virions. For illustrative purposes, the humoral response disclosed herein is measured by serum levels of anti-AAV8 antibodies (e.g., IgG) after a subject or animal, e.g., a mouse, is injected with either a rational polyploid AAV 8-8-rh10 virion or an AAV8 virion. Thus, a reduced humoral response refers to the production of less AAV8 IgG when a subject or animal is administered a rational polyploid (e.g., rational haploid) AAV 8-8-rh10 virion compared to the anti-AAV8 IgG produced when an AAV8 virion is administered to the subject or animal under similar conditions. The lower anti-AAV8 IgG produced by the rational polyploid (e.g., rational haploid) AAV 8-8-rh10 makes it more suitable for use as a gene therapy regimen, requiring a second administration. For this particular example of an AAV gene therapy regimen, it is preferred to use the rational polyploid AAV 8-8-rh10 as the first administration and a subsequent or second administration with an AAV8 vector.

[0341] In some embodiments, the population of rational polyploid AAV virions allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from the AAV8 viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV rh74 serotype; the population of rational polyploid AAV 8-8-rh74 virions elicits a reduced humoral response compared to the humoral response elicited by the parental AAV8 serotype, and the repeated dosing comprises a first administration of the population of rational polyploid AAV 8-8-rh74 virions and a second administration of parental AAV serotype 8 virions. For illustrative purposes, the humoral response disclosed herein is measured by serum levels of anti-AAV8 antibodies (e.g., IgG) after a subject or animal, e.g., a mouse, is injected with either rational polyploid AAV 8-8-rh74 virions or AAV8 virions.

[0342] Thus, the reduced humoral response leads to the production of less AAV8 IgG when a subject or animal is administered rational polyploid (e.g., rational haploid) AAV 8-8-rh74 virions compared to the anti-AAV 8 IgG produced when AAV8 virions are administered to the subject or animal under similar conditions. The lower anti-AAV8 IgG produced by the rational polyploid (e.g., rational haploid) AAV 8-8-rh74 makes it more suitable for use as a gene therapy regimen requiring a second administration. For this particular example of an AAV gene therapy regimen, it is preferred to use the rational polyploid AAV 8-8-rh74 as the first administration and a subsequent or second administration of an AAV8 vector, or conversely, an AAV8 vector as the first administration and a subsequent or second administration of the rational polyploid AAV 8-8-rh74.

[0343] As used herein, AAV vector and AAV virion are used interchangeably.

[0344] In some embodiments, a population of rational polyploid AAV virions that allows for repeated dosing comprises at least one of AAV VP1 or VP2 viral structural protein and AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from the AAV8 viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV rh10 serotype or the AAV rh74 serotype; the population of rational polyploid AAV 8-8-rh10 virions or AAV 8-8-rh74 virions elicits a reduced humoral response compared to the humoral response elicited by the parental AAV8 serotype, and the repeated dosing comprises a first administration of the parental AAV serotype 8 virions and a second administration of the population of rational polyploid AAV 8-8-rh10 or AAV 8-8-rh74 virions. To illustrate, in this particular example, AAV 8-8-rh10 or AAV 8-8-rh74 elicits a reduced humoral response, e.g., produces less anti-AAV8 IgG compared to that produced by AAV8 virions. This AAV gene therapy regimen, with a first administration of a parental AAV serotype, e.g., AAV8, and a second administration of a rational polyploid (e.g., AAV 8-8-rh10 or AAV 8-8-rh74), is suitable for achieving efficient transduction with the second administration of the rational polyploid (e.g., AAV 8-8-rh10 or AAV 8-8-rh74). As shown in Figure 26, the first administration with AAV8 produces anti-AAV8 IgG in the serum that can neutralize or inhibit AAV 8-mediated transduction, whereas AAV 8-8-rh10 or AAV 8-8-rh74-mediated transduction is not inhibited in the presence of serum containing anti-AAV 8 IgG, supporting the preferred use of AAV 8-8-rh10 or AAV 8-8-rh74 as the second administration when the first administration is with AAV 8.

[0345] In some embodiments, the population of rational polyploid AAV virions allows for repeated dosing, the population comprising at least one of AAV VP1 or VP2 viral structural protein and AAV VP3 viral structural protein; the VP1 and VP2 viral structural proteins are each from any AAV viral serotype, and the VP3 viral structural protein is selected from the rhesus AAV serotype; the population of rational polyploid AAV virions elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and the VP1 and VP2 are not from the rhesus AAV serotype; and the repeated dosing comprises a first administration of the VP1 structural viral protein or the parent AAV serotype of the VP2 structural viral protein, and a second administration of the population of rational polyploid AAV virions.

[0346] In some embodiments, a method for repeated dosing comprising first and second administrations, wherein the repeated dosing comprises a first administration of a parent AAV serotype of a VP1 or VP2 viral structural protein and a second administration of rational polyploid AAV virions, wherein the VP3 viral structural protein of the rational polyploid virions is from an AAV serotype that is different from the serotype of at least one of the VP1 or VP2 viral structural proteins that efficiently crosses the blood-brain barrier, and wherein the population of rational polyploid virions elicits a reduced humoral response compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein, and wherein VP1 or VP2 is not from a Rhesus AAV serotype.

[0347] It will be understood by those skilled in the art that the modified capsid proteins, viral capsids, viral vectors and AAV particles of the present invention exclude capsid proteins, capsids, viral vectors and AAV particles as they exist or are found in their native state. VII. Methods for Producing Viral Vectors

[0348] The AAV rational vector disclosed herein can be produced by any means known in the art.As a merely illustrative example, using AAV haploid virions as an illustrative example, the method includes: (a) transfecting host cells with one or more plasmids that together provide all the functions and genes required to assemble AAV haploid particles; (b) introducing one or more nucleic acid constructs into packaging or producer cell lines to provide all the functions and genes required to assemble AAV haploid particles; (c) introducing one or more recombinant baculovirus vectors that together provide all the functions and genes required to assemble AAV haploid particles into host cells; and / or (d) introducing one or more minicircles into host cells, or using closed linear DNA (clDNA) or barbell DNA, that together provide all the functions and genes required to assemble AAV haploid particles.

[0349] The present disclosure further provides a method for producing the rational polyploid or haploid AAV vectors disclosed herein as AAV particles. Accordingly, the present invention provides a method for producing AAV haploid virion particles comprising the rational polyploid AAV vectors of the present invention, comprising the steps of: (a) transfecting a host cell with one or more plasmids that together provide all the functions and genes required to assemble an AAV haploid vector particle; (b) introducing one or more nucleic acid constructs into a packaging or producer cell line to provide all the functions and genes required to assemble an AAV particle; (c) introducing into the host cell one or more recombinant baculovirus vectors that together provide all the functions and genes required to assemble an AAV particle; and / or (d) introducing into the host cell one or more recombinant herpesvirus vectors that together provide all the functions and genes required to assemble an AAV particle. Conditions for the formation of AAV haploid virions are standard conditions for the production of AAV vectors in cells (e.g., mammalian or insect cells), including, by way of non-limiting example, transfection of cells in the presence of an Ad helper plasmid, or other helper virus, such as HSV.

[0350] Non-limiting examples of various methods for producing the viral vectors of the invention are described in Clement and Grieger ("Manufacturing of recombinant adeno-associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev. 3:16002 (2016)) and Grieger et al. ("Production of recombinant adeno-associated viral vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector" Mol Ther 24(2):287-297 (2016)), the entire contents of which are incorporated herein by reference.

[0351] In one representative embodiment, this technology also provides a method for producing a viral vector, comprising providing a cell with (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence) and (b) AAV sequences sufficient for replication of the nucleic acid template and encapsidation into an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding an AAV capsid of the present invention). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In certain embodiments, the nucleic acid template comprises two AAV ITR sequences, which are located 5' and 3' to the heterologous nucleic acid sequence (if present), but need not be directly contiguous with the heterologous nucleic acid sequence.

[0352] The nucleic acid template and AAV rep and cap sequences are provided under conditions such that a viral vector containing the nucleic acid template packaged in an AAV capsid is produced in the cell. The method can further include a step of collecting the viral vector from the cell. The viral vector can be collected from the culture medium and / or by lysing the cell.

[0353] In one embodiment, and as disclosed herein in the Examples, the nucleic acid template is modified such that the capsid (Cap) sequence is incapable of expressing all three viral structural proteins, VP1, VP2, and VP3, from a nucleic acid sequence from only one serotype (the first nucleic acid sequence). This modification can be, for example, by eliminating the start codon for at least one of the viral structural proteins. The template will also contain at least one additional nucleic acid sequence (the second nucleic acid sequence) from a different serotype that encodes and is capable of expressing a viral structural protein that cannot be expressed by the first nucleic acid sequence, and the second nucleic acid sequence is incapable of expressing a viral structural protein that can be expressed by the first nucleic acid sequence. In one embodiment, the first nucleic acid sequence is capable of expressing two of the viral structural proteins, while the second nucleic acid sequence is capable of expressing only the remaining viral sequence. For example, a first nucleic acid sequence can express VP1 and VP2 from one serotype but cannot express VP3, and a second nucleic acid sequence can express VP3 from an alternative serotype but cannot express either VP1 or VP2. The template cannot express any other of the three viral structural proteins. In one embodiment, the first nucleic acid sequence can only express one of the three viral structural proteins, and the second nucleic acid sequence can express only the other two viral sequences but cannot express the first.

[0354] In another embodiment, there is a third nucleic acid sequence from a third serotype, in which each of the three nucleic acid sequences is only capable of expressing one of the three capsid viral structural proteins, VP1, VP2, and VP3, and each does not express the viral structural protein expressed by another of these sequences, such that collectively, capsids containing VP1, VP2, and VP3 are produced, and each of the viral structural proteins within the capsid are all only from the same serotype, and in this embodiment, VP1, VP2, and VP3 are all from different serotypes.

[0355] The modification to prevent expression can be by any means known in the art. For example, it can be by eliminating the start codon, splice acceptor, splice donor, or a combination thereof. Deletion and addition, as well as site-specific alteration to change the reading frame, can be used. The nucleic acid sequence can also be produced synthetically. These helper templates typically do not contain ITRs.

[0356] The cell can be the cell that can replicate AAV virus.Can utilize any suitable cell known in the art.In certain embodiments, the cell is a mammalian cell.Alternatively, the cell can be a trans-complementing packaging cell line that provides the function that is missing from replication-defective helper virus, such as 293 cell or other E1a trans-complementing cell.

[0357] AAV replication and capsid sequences can be provided by any method known in the art. Current protocols generally express the AAV rep / cap genes on a single plasmid. It is not necessary to provide the AAV replication and packaging sequences together, although it may be convenient to do so. The AAV rep and / or cap sequences can be provided by any viral or non-viral vector. For example, the rep / cap sequences can be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus vector). EBV vectors can also be used to express the AAV cap and rep genes. One advantage of this method is that the EBV vector is episomal but will maintain high copy number through successive cell divisions (i.e., stably integrated into cells as an extrachromosomal element termed an "EBV-based intranuclear episome"; see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

[0358] As a further alternative, the rep / cap sequences can be stably integrated into cells. Typically, the AAV rep / cap sequences will not be flanked by terminal repeats (TRs) to prevent rescue and / or packaging of these sequences.

[0359] Any method known in the art can be used to provide a nucleic acid template to a cell. For example, the template can be provided by a non-viral (e.g., a plasmid) or viral vector. In certain embodiments, the nucleic acid template is provided by a herpesvirus or adenovirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). As another example, Palombo et al., (1998) J. Virology 72:5025, describes a baculovirus vector carrying a reporter gene flanked by AAV TRs. Templates can also be delivered using EBV vectors, as described above with respect to the rep / cap gene.

[0360] In another exemplary embodiment, the nucleic acid template is provided by replicating rAAV virus. In yet other embodiments, the AAV provirus containing the nucleic acid template is stably integrated into a chromosome of the cell.

[0361] To improve virus titer, cells can be provided with helper virus functions (such as adenovirus or herpesvirus) that promote productive AAV infection.The helper virus sequences required for AAV replication are known in the art.Typically, these sequences will be provided by helper adenovirus or herpesvirus vector.Alternatively, adenovirus or herpesvirus sequences can be provided by other non-viral or viral vectors, such as the non-infectious adenovirus miniplasmid that carries all of the helper genes that promote efficient AAV production, as described by Ferrari et al., (1997) Nature Med.3:1295, and U.S. Patent No. 6,040,183 and U.S. Patent No. 6,093,570.

[0362] Additionally, helper virus functions can be provided by packaging cells in which the helper sequences are chromosomally embedded or maintained as stable extracellular elements. Generally, the helper virus sequences are not packaged into AAV virions and are not flanked by, for example, TRs.

[0363] Those skilled in the art will appreciate that it is advantageous to provide AAV replication and capsid sequences and helper virus sequences (e.g., adenovirus sequences) in a single helper construct. This helper construct can be a non-viral or viral construct. As a non-limiting example, the helper construct can be a hybrid adenovirus or hybrid herpesvirus containing the AAV rep / cap genes.

[0364] In one specific embodiment, the AAV rep / cap sequence and the adenovirus helper sequence are provided by a single adenovirus helper vector. This vector can further comprise a nucleic acid template. The AAV rep / cap sequence and / or the rAAV template can be inserted into a deleted region of the adenovirus (e.g., the E1a or E3 region).

[0365] In a further embodiment, the AAV rep / cap sequences and adenovirus helper sequences are supplied by a single adenovirus helper vector. According to this example, the rAAV template can be provided as a plasmid template.

[0366] In another illustrative embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained in the cell as an extrachromosomal element (e.g., as an EBV-based intranuclear episome).

[0367] In another exemplary embodiment, AAV rep / cap sequence and adenovirus helper sequence are provided by a single adenovirus helper.rAAV template can be provided as another replicating virus vector.For example, rAAV template can be provided by rAAV particle or second recombinant adenovirus particle.

[0368] According to the above-mentioned method, the hybrid adenovirus vector typically contains sufficient adenovirus 5' and 3' cis sequences (i.e., adenovirus terminal repeat and PAC sequences) for adenovirus replication and packaging. The AAV rep / cap sequence, and if present, the rAAV template, are embedded in the adenovirus backbone and adjacent to the 5' and 3' cis sequences, so that these sequences can be packaged into adenovirus capsids. As mentioned above, the adenovirus helper sequence and the AAV rep / cap sequence are generally not adjacent to the TR, and therefore, these sequences are not packaged into AAV virions. Zhang et al. ((2001) Gene Ther. 18:704-12) describes a chimeric helper containing both adenovirus and AAV rep and cap genes.

[0369] Herpesvirus can also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding AAV Rep proteins can advantageously facilitate scalable AAV vector production schemes.Hybrid herpes simplex virus type I (HSV-1) expressing AAV-2 rep and cap genes has been described (Conway et al., (1999) Gene Therapy 6:986 and WO00 / 17377).

[0370] As a further alternative, the AAV haploid vectors of the present invention can be produced in insect cells using a baculovirus vector to deliver the rep / cap genes and rAAV template, as described, for example, by Urabe et al., (2002) Human Gene Therapy 13:1935-43.

[0371] AAV haploid vector stocks free of contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV haploid vectors can also be separated from helper viruses based on affinity for heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973). A deletion replication-defective helper virus can be used so that any contaminating helper viruses are not replicable. As a further alternative, adenovirus helpers lacking late gene expression can be utilized, since only adenovirus early gene expression is required to mediate AAV viral packaging. Adenovirus mutants lacking late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).

[0372] In some embodiments, the methods for generating rAAVV haploid vectors disclosed herein can use rAAV producer cell lines according to the methods described in U.S. Patent No. 9,441,206, the entire contents of which are incorporated herein by reference. In particular, AAV haploid vectors or rAAV virions are produced using a method comprising: (a) providing an AAV haploid vector expression system to an rAAV producer cell line; (b) culturing the cells under conditions in which AAV haploid particles are produced; and (c) optionally isolating the AAV haploid vector particles. The triple transfection ratio of plasmids and the volume of the transfection cocktail can be optimized by varying the ratio of the XX680, AAV rep / cap helper, and TR plasmids to determine the optimal plasmid ratio for rAAV vector production.

[0373] In some examples, cells are cultured in suspension under conditions that produce AAV8 haploid particles. In another embodiment, cells are cultured under animal-derived component-free conditions. The animal-derived component-free medium can be any animal-derived component-free medium (e.g., serum-free medium) compatible with the rAAV producer cell line. Examples include, but are not limited to, SFM4Transfx-293 (Hyclone), Ex-Cell 293 (JRH Biosciences), LC-SFM (Invitrogen), and Pro10 cells, or Pro293-S (Lonza). Conditions sufficient for replication and packaging of AAV particles can be, for example, the presence of AAV sequences (e.g., AAV rep and AAV cap sequences) sufficient for replication and encapsidation of the rAAV genome into AAV capsids as described herein, as well as helper sequences from adenovirus and / or herpesvirus.

[0374] Bacterial DNA sequences from the plasmid backbone can be inadvertently packaged into AAV8 haploid capsids during the production of recombinant AAV vectors, resulting in activation of the innate immune system through their interaction with TLR9 (Akira, 2006; Chadeuf, 2005; Wright, 2014). Therefore, in some embodiments, various techniques can be used to eliminate plasmid backbone sequences in recombinant AAV haploid preparations, such as minicircles (Schnodt, 2016), which have limited scalability. Another method for avoiding bacterial DNA sequences in the plasmid backbone is the use of closed-end linear double-stranded DNA, including various DNA replication technologies, including, but not limited to, Doggybone DNA (dbDNA™), for the specific production of recombinant AAV vectors. The use of closed-end linear double-stranded DNA, such as dbDNA™, which eliminates the bacterial backbone and has been used to produce vaccines and lentiviruses (Walters et al., 2014; Scott et al., 2015; Karda et al., 2019), has been shown by DNA vaccine developers to be unable to induce TLR9 responses.

[0375] Thus, in an alternative embodiment, generation of rational polyploid or haploid AAV vectors disclosed herein, as exemplified, for example, by the production of AAV8-8-rh10 or AAV8-8-rh74 haploids, can be carried out using closed-end linear double-stranded DNA, including but not limited to barbell DNA, as disclosed in U.S. Patent Application Publication No. 2018 / 0037943 and Karbowniczek et al., Bioinsights, 2017, both of which are incorporated by reference in their entireties. Briefly, plasmids for AAV production using closed-end linear double-stranded DNA technology contain ITRs, a promoter, and a gene of interest flanked by 56-bp palindromic protelomerase recognition sequences. The plasmid is denatured, and in the presence of Phi29 DNA polymerase and appropriate primers, Phi29 initiates rolling circle amplification (RCA), resulting in the creation of double-stranded concatemeric repeats of the original construct. Addition of protelomerase results in ligation of palindromic protelomerase recognition sequences, resulting in a cleavage-join reaction that generates a monomeric, double-stranded (ds), linear, covalently closed DNA construct. Addition of common restriction enzymes removes unwanted DNA plasmid backbone sequences and digests with exonuclease activity, resulting in dbDNA, which can be size-fractionated to isolate dbDNA sequences encoding the ITRs, promoter, and gene of interest. An exemplary plasmid for generation of rAAV vectors using end-closed linear double-stranded DNA, such as dbDNA™ technology, contains the following in 5' to 3' orientation: 5'-protelomerase RS, 5' ITR, LSP promoter, hGAA, 3' UTR, hGH poly(A), 3' ITR, 3'-protelomerase RS (sense strand), where the sense strand is linked to the complementary antisense strand for a docked (ds), linear, covalently closed DNA construct.The use of closed-end, linear, double-stranded DNA, such as Doggybone DNA (dbDNA™), as the starting material for producing AAV vectors for use in the methods and compositions disclosed herein eliminates the bacterial backbone used to propagate the AAV vector-containing plasmid, the product of which is unable to elicit a Toll-like receptor 9 (TLR9) response.

[0376] Additional methods of making AAV particles are known in the art and are described in, e.g., U.S. Patent Nos. 6,204,059, 5,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526, Nos. 7291498 and 7491508, 5064764, 6194191, 6566118, and 8137948; or International Publication Nos. WO1996039530, WO1998010088, WO1999014354, WO1999015685, WO1999047691, W02000055342, W02000075353, and W02001023597; Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al., Vir., 219:37-44 (1996); Zhao et al., Vir. 272: 382-93 (2000), the contents of each of which are incorporated herein by reference in their entirety. In certain embodiments, the AAV particles are produced using the methods described in International Patent Publication WO2015191508, the contents of which are incorporated herein by reference in their entirety.

[0377] Virus replication cells can be selected from any biological organism, including prokaryotic (for example, bacterial) cells and eukaryotic cells, including insect cells, yeast cells and mammalian cells.Virus replication cells commonly used for producing recombinant AAV virus particles include but are not limited to HEK293 cells, COS cells, HeLa cells, KB cells and other mammalian cell lines, such as those described in U.S. Patent No. 6,156,303, U.S. Patent No. 5,387,484, U.S. Patent No. 5,741,683, U.S. Patent No. 5,691,176 and U.S. Patent No. 5,688,676, U.S. Patent Application Publication No. 2002 / 0081721, and International Patent Application No. WO2000047757, WO2000024916 and WO1996017947, and the contents of each of these reference patent documents are incorporated herein by reference in their entirety. Viral-replicating cells can include other mammalian cells, such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, Saos, C2C12, L cells, HT1080, HepG2, as well as primary fibroblasts, hepatocytes, and myoblasts derived from mammalian species, including, but not limited to, humans, monkeys, mice, rats, rabbits, and hamsters. Viral-replicating cells can include cells derived from cell types, including, but not limited to, fibroblasts, hepatocytes, tumor cells, cell line-transformed cells, and the like. VIII. Recombinant AAV Polyploid Virus Vectors

[0378] The present invention provides a method of administering a nucleic acid molecule to a cell, the method comprising contacting the cell with a rAVV haploid viral vector and / or composition or pharmaceutical formulation of the present invention.

[0379] The present invention further provides a method for delivering nucleic acid to a subject, the method comprising the step of administering to a subject an AAV haploid viral vector, AAV particle and / or composition or pharmaceutical formulation of the present invention.

[0380] In certain embodiments, the subject is human, and in some embodiments, the subject has or is at risk for a disorder that can be treated by a gene therapy protocol. Non-limiting examples of such disorders include neurological disorders, including but not limited to epilepsy, depression, Huntington's disease, Parkinson's disease or Alzheimer's disease, ADHD, ASD, autoimmune diseases, cystic fibrosis, thalassemia, Hurler syndrome, Sly syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteaux-Lamy syndrome, Krabbe disease, phenylketonuria, Batten disease, and spinal cerebral ataxia; LDL receptor deficiency; hyperammonemia; anemia; arthritis; retinal degenerative disorders, including macular degeneration; adenosine deaminase deficiency; metabolic disorders; and cancer, including oncogenic cancer.

[0381] In some embodiments of the methods of the present invention, the rAVV haploid vector and / or composition or pharmaceutical formulation of the present invention may be administered to skeletal muscle, cardiac muscle and / or diaphragm muscle.

[0382] In the methods described herein, the rAVV haploid vectors and / or compositions or pharmaceutical formulations of the present invention can be administered / delivered to a subject of the present invention by a systemic route (e.g., intravenously, intraarterially, intraperitoneally, etc.). In some embodiments, the viral vectors and / or compositions can be administered to a subject by intracerebroventricular, intracisternal, intraparenchymal, intracranial, and / or intrathecal routes. In certain embodiments, the rational polyploid or haploid AAV vectors disclosed herein and / or pharmaceutical formulations of the present invention are administered intrathecally or intravenously.

[0383] The rAVV haploid vectors disclosed herein are useful for delivering nucleic acid molecules to cells in vitro, ex vivo, and in vivo. In particular, rAVV haploid vectors can be advantageously used to deliver or transfer nucleic acid molecules to animal cells, including mammalian cells.

[0384] Any heterologous nucleic acid sequence of interest can be delivered in the rAVV haploid vectors of the invention. Nucleic acid molecules of interest include nucleic acid molecules encoding polypeptides, including therapeutic polypeptides (e.g., for medical or veterinary use) and / or immunological polypeptides (e.g., for vaccines).

[0385] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin (including mini-dystrophins and micro-dystrophins; see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Application Publication No. 2003 / 017131; International Patent Publication No. WO / 2008 / 088895; Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as IkappaB dominant variants, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ, interferon-γ), These proteins include: leukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial cell line-derived growth factor, transforming growth factor-α and -β, etc.), lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis factor-α soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, and molecules that modulate calcium handling (e.g., SERCA). 2A, inhibitors of PP1 and fragments thereof [e.g., WO2006 / 029319 and WO2007 / 100465]), molecules that cause G protein-coupled receptor kinase type 2 knockdown, such as truncated constitutively active bARKct, anti-inflammatory factors such as IRAP, anti-myostatin protein, aspartoacylase, monoclonal antibodies (including single-chain monoclonal antibodies; an exemplary Mab is Herceptin® Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see, e.g., U.S. Pat. No. 7,071,172)), angiogenesis inhibitors, such as vasohibin and other VEGF inhibitors (e.g., vasohibin 2 [see WO JP2006 / 073052]). Other illustrative heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer treatment, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptides that have a therapeutic effect in a subject in need thereof. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, such as antibodies or antibody fragments against myostatin (see, e.g., Fang et al., Nature Biotechnology 23:584-590 (2005)).

[0386] Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein (GFP), luciferase, β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase genes.

[0387] Optionally, the heterologous nucleic acid molecule encodes a secreted polypeptide (e.g., a polypeptide that is a secreted polypeptide in its native state or a polypeptide that has been engineered to be secreted, e.g., by operable association with a secretory signal sequence as known in the art).

[0388] Alternatively, in certain embodiments of the invention, the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that causes spliceosome-mediated trans-splicing (see, e.g., Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. Nos. 6,013,487, 6,083,702), an interfering RNA (RNAi) including siRNA, shRNA, or miRNA that mediate gene silencing (see, Sharp et al., (2000) Science 287:2431), and other non-translated RNA, such as a "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.).

[0389] In one embodiment, the rational polyploid vector comprises a viral genome encoding an RNAi agent payload, for example, the RNAi agent may be, but is not limited to, dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA. When expressed, the RNAi agent inhibits or suppresses the expression of a gene of interest in a cell, for example, but is not limited to, SOD1, MAPT, APOE, HTT, C90RF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN2, ATXN3, ATXN7, SCN1A-SCN5A, or SCN8A-SCN11A.

[0390] In one embodiment, the rational polyploid vector disclosed herein comprises a viral genome encoding a polypeptide payload. The polypeptide can be, but is not limited to, an antibody, aromatic L-amino acid decarboxylase (AADC), survival motor neuron 1 (SMN1), frataxin (FXN), APOE (APOE2, APOE3, or APOE4), GBA1, GRN, ASP A, CLN2, GLB1, SGSH, NAGLU, IDS, NPC1, or GAN.

[0391] Exemplary non-coding RNAs include RNAi against multidrug resistance (MDR) gene products (e.g., for administration to the heart to treat and / or prevent tumors and / or to prevent chemotherapy damage); RNAi against myostatin (e.g., for Dusenne muscular dystrophy); RNAi against VEGF (e.g., for treating and / or preventing tumors); RNAi against phospholamban (e.g., for treating cardiovascular disease; see, e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory or dominant-negative molecules, such as phospholamban S16E (e.g., for treating cardiovascular disease; see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2005)). (2002)), RNAi against adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0392] Furthermore, nucleic acid sequences that direct alternative splicing can be delivered. For example, an antisense sequence (or other inhibitory sequence) complementary to the 5' and / or 3' splice site of the 51st exon of dystrophin can be delivered together with a U1 or U7 small nuclear (sn)RNA promoter to induce skipping of this exon. For example, a DNA sequence containing a U1 or U7 snRNA promoter located 5' from the antisense / inhibitory sequence can be packaged in the modified capsid of the present invention and delivered.

[0393] Viral vectors can also contain heterologous nucleic acid molecules that share homology with and recombine with loci on host cell chromosomes. This approach can be used, for example, to correct genetic defects in the host cell.

[0394] The present invention also provides rAVV haploid vectors expressing immunogenic polypeptides, peptides, and / or epitopes, e.g., for vaccination. The nucleic acid molecules can encode any immunogen of interest known in the art, including, but not limited to, immunogens from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc.

[0395] The use of parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Patent No. 5,916,563 to Young et al.; U.S. Patent No. 5,905,040 to Mazzara et al.; U.S. Patent No. 5,882,652 to Samulski et al.; and U.S. Patent No. 5,863,541 to Samulski et al.). Antigens can be presented within the parvovirus capsid. Alternatively, immunogens or antigens can be expressed from heterologous nucleic acid molecules introduced into the recombinant vector genome. Any immunogen or antigen of interest, as described herein and / or known in the art, can be provided by the viral vectors of the present invention.

[0396] Immunogenic polypeptides for delivery by rAAV haploid vectors for use as vaccines disclosed herein can be any polypeptide, peptide, and / or epitope suitable for eliciting an immune response against and / or protecting a subject from infections and / or diseases, including, but not limited to, microbial, bacterial, protozoan, parasitic, fungal, and / or viral infections and diseases. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as an HIV or SIV envelope GP160 protein, an HIV or SW matrix / capsid protein, and HIV or SIV gag, pol, and env gene products). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a Lassa virus immunogen, e.g., a Lassa virus nucleocapsid protein and a Lassa virus envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, e.g., a vaccinia L1 or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, e.g., the NP and GP gene products), a bunyavirus immunogen (e.g., an RVFV, CCHF, and / or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, e.g., a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen).The immunogenic polypeptide may further be a polio immunogen, a herpes immunogen (e.g., a CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or any other vaccine immunogen now known in the art or later identified as an immunogen.

[0397] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in SA Rosenberg (Immunity 10:281 (1991)).Other illustrative cancer and tumor antigens include, but are not limited to, the following: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124), MART-1, gp100. MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2 / neu gene product (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl-Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigens (International Patent Publication No. WO 90 / 05142); telomerase; nuclear matrix proteins; prostatic acid phosphatase; papillomavirus antigens; and / or antigens now known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

[0398] As a further alternative, the heterologous nucleic acid molecule can encode any polypeptide, peptide, and / or epitope that is desirably produced in a cell in vitro, ex vivo, or in vivo. For example, an AAV haploid vector can be introduced into cultured cells and the expressed gene products isolated therefrom.

[0399] Those skilled in the art will understand that a heterologous nucleic acid molecule of interest can be operably associated with appropriate control sequences. For example, the heterologous nucleic acid molecule can be operably associated with expression control elements, such as transcriptional / translational control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and / or enhancers.

[0400] Furthermore, regulated expression of a heterologous nucleic acid molecule of interest can be achieved at the post-transcriptional level by modulating alternative splicing of different introns, for example, by the presence or absence of oligonucleotides, small molecules and / or other compounds that selectively block splicing activity at specific sites (e.g., as described in WO2006 / 119137, which is incorporated herein by reference).

[0401] Those skilled in the art will understand that various promoter / enhancer elements can be used depending on the desired level and tissue-specific expression. The promoter / enhancer can be constitutive or inducible, depending on the desired pattern of expression. The promoter / enhancer can be native or foreign, and can be a natural sequence or a synthetic sequence. By foreign, it is meant that the transcription initiation region is not found in the wild-type host into which it is introduced.

[0402] In certain embodiments, the promoter / enhancer element may be native to the target cell or subject to be treated. In representative embodiments, the promoter / enhancer element may be native to the heterologous nucleic acid sequence. In some embodiments, the promoter sequence or regulatory sequence is a CNS-specific promoter. In some embodiments, the CNS-specific promoter is disclosed in UK Patent Application GB 2007539.6, the entire contents of which are incorporated herein by reference. The promoter / enhancer element is generally selected so that it functions in the target cell of interest, e.g., CNS tissue, including neural and non-neuronal cells in the CNS or PNS. Furthermore, in certain embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0403] Inducible expression control elements are generally advantageous in applications where it is desirable to regulate the expression of heterologous nucleic acid sequences. Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferential promoter / enhancer elements, including muscle-specific or preferential (including cardiac, skeletal, and / or smooth muscle-specific or preferential), nervous tissue-specific or preferential (including brain-specific or preferential), eye-specific or preferential (including retina-specific or cornea-specific), liver-specific or preferential, bone marrow-specific or preferential, pancreas-specific or preferential, spleen-specific or preferential, and lung-specific or preferential promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters.

[0404] In embodiments in which the heterologous nucleic acid sequence is transcribed in the target cell and then translated therein, specific initiation signals are generally included for efficient translation of the inserted protein-coding sequence. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0405] The AAV haploid vector of the present invention provides a means for delivering heterologous nucleic acid molecules to a wide range of cells, including dividing and non-dividing cells. AAV haploid vectors can be used to deliver nucleic acid molecules of interest to cells in vitro, for example, for producing polypeptides in vitro or for ex vivo or in vivo gene therapy. Viral vectors can also be useful in methods for delivering nucleic acids to subjects in need thereof, for example, to express immunogenic or therapeutic polypeptides or functional RNA. In this way, polypeptides or functional RNAs can be produced in vivo in subjects. The subject may need a polypeptide because the subject is deficient in the polypeptide.

[0406] Additionally, the methods can be practiced because production of a polypeptide or functional RNA in a subject can have some beneficial effect.

[0407] Viral vectors can also be used to produce a polypeptide or functional RNA of interest in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce a polypeptide, or using the subject to observe the effect of a functional RNA on the subject, e.g., in connection with a screening method).

[0408] Generally, the viral vectors of the present invention can be utilized to deliver heterologous nucleic acid molecules encoding polypeptides or functional RNA to treat and / or prevent any disorder or condition in which delivery of a therapeutic polypeptide or functional RNA is beneficial. Illustrative disease states include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane conductance regulator) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (endostatin; angiostatin; TRAIL; FAS-ligand; cytokines including interferons; R for VEGF). RNAi, including RNAi; or multidrug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma]), diabetes (insulin); Dusenne muscular dystrophies, including RNAi against dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., alpha, beta, gamma], myostatin, myostatin propeptide, follistatin, activin type II soluble receptors, anti-inflammatory polypeptides, e.g., IκB dominant mutants, sarcospan, utrophin, mini-utrophin, antisense to splice junctions in the dystrophin gene to induce exon skipping [see, e.g., WO2003 / 095647], antisense to U7 snRNA to induce exon skipping [see, e.g., WO2006 / 021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker muscular dystrophies;Gaucher disease (glucocerebrosidase), Hurler disease (α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (Schiff disease) vasohibin or other inhibitors of VEGF or other angiogenesis inhibitors to treat / prevent retinal disorders, e.g., retinal degeneration (as well as other diseases of the eye and retina; e.g., PDGF for macular degeneration, and / or retinal disorders, e.g., retinal disorders in type 1 diabetes), diseases of solid organs, e.g., diseases of the brain (Parkinson et al., 2004). Cancer disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin, and / or VEGF], glioblastoma [RNAi against endostatin, angiostatin, and / or VEGF]), liver disease, kidney disease, cardiac disease including congestive heart failure or peripheral arterial disease (PAD) (e.g., protein phosphatase inhibitor I (I-1) and its fragments (e.g., I1C), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that result in G protein-coupled receptor kinase type 2 knockdown, such as truncated constitutively active bARKct; calsarcin, RNAi against phospholamban;and phospholamban inhibitors or dominant-negative molecules, such as phospholamban S16E, delivered by these molecules. These include arthritis (insulin-like growth factors), joint disorders (insulin-like growth factors 1 and / or 2), intimal hyperplasia (e.g., by delivering enos, inos), improved heart transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, e.g., IRAP and TNFα soluble receptor), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease; spinal and cerebral ataxias, including SCA1, SCA2, and SCA3; phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. Additionally, following organ transplantation, the present invention can be used to increase the success rate of the transplant (e.g., by administering immunosuppressants or inhibitory nucleic acids to block cytokine production) and / or reduce the negative side effects of organ transplantation or adjuvant therapy. As another example, bone morphogenetic proteins (including BNP2, 7, etc., RANKL and / or VEGF) can be administered with bone allografts, for example, after fracture or surgical removal in cancer patients.

[0409] The present invention can also be used to produce induced pluripotent stem cells (iPS). For example, the AVV haploid vector of the present invention can be used to deliver stem cell-related nucleic acids to non-pluripotent cells, such as adult fibroblasts, skin cells, liver cells, kidney cells, adipocytes, cardiac cells, neural cells, epithelial cells, endothelial cells, etc. Nucleic acids encoding stem cell-related factors are known in the art. Non-limiting examples of such factors related to stem cells and pluripotency include Oct-3 / 4, SOX family (e.g., SOX1, SOX2, SOX3, and / or SOX15), Klf family (e.g., Klf1, Klf2, Klf4, and / or Klf5), Myc family (e.g., C-myc, L-myc, and / or N-myc), NANOG, and / or LIN28.

[0410] The present invention can be practiced to treat metabolic disorders, such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), lysosomal storage disorders, such as mucopolysaccharidosis disorders (e.g., Sly syndrome [β-glucuronidase], Hurler syndrome [α-L-iduronidase], Scheie syndrome [α-L-iduronidase], Hurler-Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronate sulfatase], Sanfilippo syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA; α-glucosaminide acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfatase], B [β-galactosidase], Maroteaux-Lamy syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (e.g., Pompe disease; lysosomal acid α-glucosidase).

[0411] Gene transfer has considerable potential for use in understanding and administering treatments for disease states. There are several genetic diseases for which the defective genes are known and have been cloned. Generally, these disease states fall into two classes: deficiency states, usually enzyme deficiency states, which are generally inherited in a recessive manner; and imbalance states, which may involve regulatory or structural proteins and are typically inherited in a dominant manner. For deficiency state diseases, gene transfer can be used to introduce normal genes into affected tissues for replacement therapy, as well as to create animal models of the disease using antisense mutations. For imbalance disease states, gene transfer can be used to produce the disease state in a model system, which can then be used to attempt to combat the condition. Thus, the AAV haploid vector of the present invention enables the treatment and / or prevention of genetic diseases.

[0412] The AVV haploid vector of the present invention can also be used to provide functional RNA to cells in vitro or in vivo. Expression of functional RNA in cells can, for example, reduce the expression of a specific target protein by the cell. Thus, functional RNA can be administered to reduce the expression of a specific protein in a subject in need thereof. Functional RNA can also be administered to cells in vitro to regulate gene expression and / or cellular physiology, for example, to optimize cell or tissue culture systems or in screening methods.

[0413] In addition, the AAV haploid vectors according to the invention are used in diagnostic and screening methods in which a nucleic acid of interest is expressed transiently or stably in cell culture systems or alternat...

Claims

1. A composition suitable for use in crossing the blood-brain barrier, comprising a population of rational polyploid AAV virions, said rational polyploid AAV virions comprising at least one of AAV VP1 or VP2 viral structural proteins and an AAV VP3 viral structural protein; said at least one of VP1 or VP2 viral structural proteins is each from any AAV serotype, and said VP3 viral structural protein is from an AAV serotype different from said serotype of at least one of VP1 or VP2 that efficiently crosses the blood-brain barrier; the composition is capable of crossing the blood-brain barrier (BBB) ​​upon systemic or intrathecal administration and / or of transducing endothelial cells of the BBB and / or blood components that cross the BBB; composition.

2. The population exhibits enhanced transduction activity across the blood-brain barrier (BBB) ​​compared to non-rational polyploid AAV particles that lack the ability to cross the blood-brain barrier; and / or said VP3 viral structural protein is an AAV rhesus serotype, and / or the VP3 viral structural protein is from a serotype that efficiently crosses the blood-brain barrier selected from the group consisting of AAV1, AAV6, AAV6.2, AAV7, AAV9, AAVrhlO, AAVrh74, AAVrh39, and AAVrh43; and / or and / or wherein administration of the composition exhibits enhanced transduction of one or more of the subject's cortex, striatum, thalamus, medulla, hippocampus, cerebellum, and spinal cord, as compared to non-rational polyploid AAV particles that lack the ability to efficiently cross the blood-brain barrier; and / or wherein administration of the composition exhibits enhanced transduction compared to AAV2 in one or more CNS regions selected from the group consisting of the medullary plexus, cervical plexus, thoracic plexus, lumbar plexus, and choroid plexus; and administration of said composition is characterized by enhanced binding to brain microvascular endothelial cells (BMVEC) compared to AAV8; and / or the population has a biodistribution in the CNS; and / or and / or the population has a CNS biodistribution of at least 0.05 vg / cell, 0.1 vg / cell, at least 0.2 vg / cell, at least 0.4 vg / cell, at least 0.6 vg / cell, at least 0.8 vg / cell, at least 1 vg / cell, at least 5 vg / cell, at least 10 vg / cell, at least 20 vg / cell, at least 25 vg / cell or preferably more; and / or said at least one of VP1 or VP2 is selected from an AAV serotype that crosses the blood-brain barrier; or said at least one of VP1 or VP2 is selected from an AAV serotype that does not cross the blood-brain barrier; and / or At least one of VP1 or VP2 is not selected from the AAV rhesus serotype; or said at least one of VP1 or VP2 is selected from the AAV rhesus serotype; and / or and / or wherein, when administered to a subject, administration of said composition elicits a humoral immune response that is reduced compared to the humoral response elicited by a parent AAV vector of said VP1 or VP2 structural protein subtype; and / or the population escapes neutralizing antibodies against parent serotypes of AAV VP1, VP2 or VP3 viral structural proteins; The composition of claim 1.

3. A composition comprising a population of rational polyploid AAV virions according to claim 1 or 2, (a) a composition for delivering a transgene across the blood-brain barrier of a subject, characterized in that the composition is administered to the subject; and / or (b) a composition for repeated dosing of an AAV to a subject, the composition being administered using a first administration by administering the composition to the subject, and a second administration by administering the at least one parent AAV serotype of a VP1 or VP2 viral structural protein to the subject; wherein administration of the composition elicits a reduced humoral response in the subject compared to the humoral response elicited by the parent AAV serotype of the VP1 or VP2 viral structural protein; said at least one of VP1 or VP2 is not from a Rhesus AAV serotype; composition.

4. A rational population of polyploid AAV virions that allows for repeated dosing, A rational polyploid AAV virion comprising at least one of AAV VP1 or VP2 viral structural proteins and an AAV VP3 viral structural protein; said at least one of VP1 or VP2 viral structural proteins is each from any AAV viral serotype, and said VP3 viral structural protein is selected from the rhesus AAV serotype; said population of rational polyploid AAV virions elicits a reduced humoral response compared to the humoral response elicited by a parental AAV serotype of said VP1 or VP2 viral structural protein; said at least one of VP1 or VP2 is not from a Rhesus AAV serotype; said repeated dosing comprising a first administration of said population of rational polyploid AAV virions and a second administration of a parental AAV serotype of said VP1 or VP2 structural viral proteins; Group.

5. A population of rational polyploid AAV virions, comprising: a. VP1 and VP2 AAV viral structural proteins selected from the AAV8 viral serotype; b. A VP3 selected from the AAV rhesus serotypes AAV rhlO or AAVrh74; Including, A population of rational polyploid AAV virions that, when administered to a subject, elicits a reduced humoral response compared to the corresponding humoral response elicited by the parental AAV8 serotype.

6. A composition comprising the composition of claim 1 or a population of rational polyploid AAV virions of claims 4 or 5 for repeated dosing, wherein said composition or said population is administered using a first and a second administration of AAV to a subject; The first administration is performed by administering the composition or the population of rational polyploid AAV virions to the subject; and the second administration is performed by administering the parent AAV serotype of a VP1 or VP2 viral structural protein; said first administration elicits a reduced humoral response in said subject compared to a corresponding humoral response elicited by said parent AAV serotype of a VP1 or VP2 viral structural protein; VP1 or VP2 is not from a Rhesus AAV serotype; composition.

7. A population described in any of claims 4 to 5, or a composition described in claim 1, wherein the population or composition evades neutralizing antibodies against parent serotypes of AAV VP1, VP2 or VP3 viral structural proteins.

8. A composition comprising a population of rational polyploid AAV virions described in any one of claims 4 to 5 or the composition described in claim 1 for delivering an introduced gene across the blood-brain barrier of a subject, characterized in that the population or the composition is administered to the subject.

9. the VP3 protein is a mutated VP3 protein from an AAVrhlO or AAVrh74 serotype, and optionally The mutated AAVrh74 VP3 protein has the amino acid sequence of SEQ ID NO:2, or the amino acid sequence of a protein having at least 85% sequence identity to SEQ ID NO:2, or said mutated AAVrh74 VP3 comprises at least one of the following modifications of SEQ ID NO:2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P, and optionally The mutant AAVrhlO VP3 protein is encoded by a nucleic acid of SEQ ID NO:5 that comprises at least one or more of Q214N, S462N and D517E mutations compared to the AAVrhlO_VP3 nucleic acid of SEQ ID NO:5, or is encoded by a nucleic acid of SEQ ID NO:5 that comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO:5 that comprises at least one mutation selected from Q214N, S462N and D517E, and optionally The VP3 protein is an AAVrh74 VP3 protein comprising the amino acid sequence of SEQ ID NO: 2 or 3, or the amino acid sequence of a protein having at least 85% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 3, or comprising at least one of the following amino acid modifications of SEQ ID NO: 2: N263S, G264A, T265S, S266T, G268A, T270del, T274H, E533K, R726H, N736P. A population according to claim 4 or 5 or a composition according to claim 1.

10. The population is at least 10 １ 6. The composition of claim 1 or the substantially homogenous population of virions of claims 4 or 5, wherein the substantially homogenous population elicits significantly less anti-AAV IgG antibodies against the parent AAV serotype of VP1 or VP2 structural proteins in serum in vivo compared to a substantially homogenous population of virions comprising a parent AAV serotype, and optionally the parent AAV serotype is AAV8.

11. In the 5' to 3' direction, a. a first nucleic acid encoding an AAVrhlO VP3 capsid protein operably linked to a first promoter; b. a first polyA sequence; c. a second nucleic acid encoding a rep protein; d. a third nucleic acid encoding AAV8 VP1 and VP2 viral structural proteins, wherein the third nucleic acid sequence is incapable of expressing AAV8 VP3 viral structural protein; and e. A second polyA sequence Contains or a. a first nucleic acid encoding an AAVrh74 VP3 capsid protein operably linked to a first promoter; b. a first polyA sequence; c. a second nucleic acid encoding a rep protein; d. a third nucleic acid encoding AAV8 VP1 and VP2 viral structural proteins, wherein the third nucleic acid sequence is incapable of expressing AAV8 VP3 viral structural protein; and e. A second polyA sequence A nucleic acid comprising:

12. a. AAV virions from the population of any of claims 4 or 5; and b. A nucleic acid comprising at least one terminal repeat sequence and a heterologous gene. Including, the nucleic acid is packaged in the AAV virion; Viral vector.

13. 6. The composition of claim 1 or the population of claims 4 or 5, comprising a chimeric or modified viral structural protein, said modified viral structural protein comprising one or more amino acid insertions, deletions or substitutions.

14. A rational population of polyploid AAV virions that allows for repeated dosing, comprising at least one of an AAV VP1 or VP2 viral structural protein and an AAV VP3 viral structural protein; said VP1 and VP2 viral structural proteins are each from any AAV viral serotype except the Rhesus AAV serotype, and said VP3 viral structural protein is selected from the Rhesus AAV serotype; said population of rational polyploid AAV virions evades neutralizing antibodies against the parent AAV rhesus serotype of said VP3 viral structural protein; said repeated dosing comprising a first administration of said parental AAV rhesus serotype of said VP3 structural protein, and a second administration of said population of rational polyploid AAV virions; The VP3 structural protein of the rational polyploid virion is the AAV rhesus mutant virus structural protein VP3, and optionally said AAV rhesus mutant viral structural protein VP3 is from a mutant AAV rhlO VP3 viral structural protein or from a mutant AAV rh74 VP3 viral structural protein; and / or the mutant viral structural protein VP3 comprises mutations at amino acids corresponding to amino acids selected from the group consisting of N263, G264, T265, S266T, G268, T270, T274, and E533, all of said amino acid positions corresponding to native VP1 sequence numbering of AAV rhlO or AAVrh74, and optionally the mutation is selected from the group consisting of N263S, G264A, T265S, S266T, G268A, T270del, T274H, and E533K; and / or the mutant viral structural protein VP3 further comprises a mutation at an amino acid corresponding to an amino acid selected from the group consisting of R727 and N737, all of said amino acid positions corresponding to the native VP1 sequence numbering of AAVrhlO, and optionally the mutation is selected from the group consisting of R727H and N737P; and / or the mutant viral structural protein VP3 further comprises a mutation at an amino acid corresponding to an amino acid selected from the group consisting of R726 and N736, all of said amino acid positions corresponding to native VP1 sequence numbering of AAV rh74, and optionally the mutation is selected from the group consisting of R726H and N736P, and optionally the mutant viral structural protein VP3 further comprises a mutation at amino acid corresponding to W at 581, said W being replaced by two subsequent V residues (VV), all of the amino acid positions corresponding to the native VP1 sequence numbering of AAV rh74; and / or the AAV VP1 or VP2 viral structural protein is any AAV serotype selected from Table 1; and / or The AAV VP1 or VP2 structural protein is AAV8. Rational population of polyploid AAV virions.

15. 6. A composition comprising the composition of claim 1 or the population of rational polyploid AAV virions of claim 4 or 5 for use in the treatment of a brain disease, or brain disorder, or a neurodegenerative disease, or a neurological disease, or for use in the treatment of a disease of the central nervous system (CNS) or peripheral nervous system (PNS), or for use in the treatment of brain cancer or a subject having brain cancer, or for use in the treatment of a subject having a disease or disorder selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and dopamine transporter deficiency syndrome.

16. A composition comprising the composition of claim 1 or the population of rational polyploid AAV virions of claims 4 or 5 for use in delivering a transgene to the small intestine or for use in treating a gastrointestinal disease or disorder.

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