Materials and methods for treating disorders associated with mutations in the IRF2BPL gene

Abstract

The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of interferon regulatory factor 2-binding protein-like (IRF2BPL) disorders.

Description

[Technical Field]

[0001] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and can be found as follows: 54846_Seqlisting.txt, Size: 43,149 bytes, Created: October 19, 2020.

[0002] The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), designed for the treatment of disorders caused by mutations in interferon regulatory factor 2-binding protein-like (IRF2BPL). The disclosed AAVs provide an intact copy of the IRF2BPL cDNA capable of producing a functional protein. [Background technology]

[0003] The interferon regulatory factor 2-binding protein-like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators. The deduced 796-amino acid protein has a calculated molecular mass of 82.7 kD. It is proline-rich and contains an N-terminal polyglutamine and polyalanine tract, a C-terminal C3HC4-type RING finger domain, and two putative transmembrane domains. It also contains a potential nuclear targeting signal, an endoplasmic reticulum retention signal, three possible PEST sequences, and putative sites for phosphorylation, N-glycosylation, and amidation. RT-PCR analysis of human tissues detected strong expression in the heart, moderate expression in skeletal muscle and pancreas, and weak expression in the brain, kidney, liver, testis, thyroid, and lymphocytes. There are two mammalian paralogs, IRF2BP1 and IRF2BP2, which share two highly conserved domains. Using DNA microarrays, Heger et al. (2007) found that expression of IRF2BPL, which they named Eap1, increased in the medial basal hypothalamus but not in the cerebral cortex of female rhesus monkeys during precocious puberty, with a further increase during mid-puberty. Female mice underwent a similar increase in Eap1 expression in the hypothalamus but not in the cortex during puberty. The function of this protein is unknown, and until recently it had not been associated with Mendelian diseases.

[0004] Mutations in the IRF2BPL gene have been found to be associated with the childhood neurodevelopmental disorder NEDAMSS (neurodevelopmental disorder with regression, abnormal movements, speech loss, and seizures). NEDAMSS is a childhood-onset, progressive disorder inherited in an autosomal dominant manner. There are currently no therapies for NEDAMSS or any other IRF2BPL disorders, and there is a need to develop such therapies. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, described herein is a polynucleotide comprising (a) one or more regulatory control elements and (b) an interferon regulatory factor 2-binding protein-like (IRF2BPL) cDNA sequence. In some embodiments, the regulatory control element is a pIRF promoter, a p546 promoter comprising the nucleotide sequence set forth in SEQ ID NO:5, or a CBA promoter comprising the nucleotide sequence set forth in SEQ ID NO:6, or a fragment thereof that retains regulatory control or promoter activity. In some embodiments, the IRF2BPL cDNA comprises the polynucleotide sequence set forth in SEQ ID NO:1.

[0006] In another aspect, the present invention provides a recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequence described herein.In some embodiments, the rAAV is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and their derivatives.

[0007] In some embodiments, the rAAV genome comprises a pIRF promoter fragment and an IRF2BPL cDNA. The nucleotide sequence of the pIRF promoter fragment is 1034 base pairs and is set forth as SEQ ID NO: 10. For example, the rAAV genome comprises the pIRF promoter comprising nucleotides 1 to 1034 of pIRF (SEQ ID NO: 10), or a promoter fragment comprising nucleotides 386 to 1034 of pIRF (SEQ ID NO: 9), or a promoter fragment comprising nucleotides 738 to 1034 of pIRF (SEQ ID NO: 8).

[0008] In some embodiments, the genome of the rAAV comprises a p546 promoter and an IRF2BPL cDNA.

[0009] In some embodiments, the genome of the rAAV comprises a CBA promoter and an IRF2BPL cDNA.

[0010] In some embodiments, the genome of the rAAV comprises a pIRF promoter fragment, a p546 promoter, and an IRF2BPL cDNA, wherein the promoter retains promoter activity.

[0011] In exemplary embodiments, the genome of the rAAV comprises nucleotides 342 to 5299 of SEQ ID NO:3, or nucleotides 342 to 5031 of SEQ ID NO:4, or nucleotides 342 to 5070 of SEQ ID NO:7.

[0012] In another aspect, described herein are rAAV particles comprising the rAAV described herein.

[0013] Particularly contemplated is a method for treating an interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder in a subject in need thereof, comprising administering the rAAV or rAAV particles described herein. An IRF2BPL-associated disorder is a neurological disorder associated with the presence of a mutation in the IRF2BPL gene. In some embodiments, the IRF2BPL-associated disorder includes NEDAMSS (neurodevelopmental disorder associated with regression, abnormal movements, speech loss, and seizures), or other neurological disorders, including, but not limited to, epilepsy, schizophrenia, and neurological diseases.

[0014] In another aspect, described herein is the use of an rAAV or rAAV particle described herein in the preparation of a medicament for the treatment of an interferon regulatory factor 2 binding protein-like (IRF2BPL)-associated disorder, such as NEDAMSS.

[0015] In another aspect, described herein are compositions comprising an rAAV or rAAV particles described herein for the treatment of an interferon regulatory factor 2 binding protein-like (IRF2BPL)-associated disorder, such as NEDAMSS. [Brief explanation of the drawings]

[0016] [Figure 1]Schematic diagram of IRF2BPL protein domains and the locations of mutations present in patient-derived skin fibroblasts used in this study. [Figure 2] Figure 2A shows IRF2BPL protein expression detected by immunofluorescence. Figure 2B shows IRF2BPL protein expression detected by Western blot (n=3). The data show that there is no significant difference in protein expression levels between healthy controls and NEDAMSS patients, except for one patient (P3) who showed a reduction. [Figure 3] A graph showing the expression of IRF2BPL protein in astrocytes (iAST) derived from fibroblasts isolated from healthy individuals and patients with NEDAMSS is provided. No significant differences in the overall levels of protein expression were observed, except for the cell line (P3) from the same patient whose fibroblasts already in Figure 2 showed reduced IRF2BPL expression. [Figure 4A] Representative images showing abnormal accumulation of IRF2BPL in the cytoplasm of iAST from patients with NEDAMSS (P1, P2, P3, and P4). The blue stain is DAPI, and the red stain is IRF2BPL; when overlapped, the staining appears purple. [Figure 4B] Representative images showing abnormal accumulation of IRF2BPL in the cytoplasm of iAST from patients with NEDAMSS (P1, P2, P3, and P4). The blue stain is DAPI, and the red stain is IRF2BPL; when overlapped, the staining appears purple. [Figure 5] A graph showing the normalized ratio of cell number to DAPI count with cytoplasmic accumulation of IRF2BPL in fibroblasts is provided (n=3). [Figure 6]We provide graphs showing IRF2BPL protein expression in nuclear (Figure 6A) and cytoplasmic (Figure 6B) extracts from patient and control astrocytes (n = 3), supporting increased cytoplasmic accumulation and decreased nuclear localization in patient iAST. [Figure 7] Representative images and quantification are provided showing mouse motor neuron viability after 3 days of co-culture of patient or healthy iAST with GFP+ motor neurons (black), n=4. [Figure 8] Immunofluorescence staining of WNT1 and DAPI in NEDAMSS patient cell lines. WNT1 expression was increased in NEDAMSS patients. [Figure 9] Western blot data measuring WNT1 levels in lysates and supernatants of AST from healthy individuals and NEDAMSS patients are provided (n=1). The graph shows that WNT1 expression is increased in the supernatants of astrocytes from NEDAMSS patients. [Figure 10A] Representative images are provided showing that the number of neurons was reduced in NEDAMSS patient cell lines compared to healthy controls after direct reprogramming from fibroblasts. Figure 10A provides staining using the pan-neuronal marker Tuj1, and Figure 10B provides staining for the neuron-specific marker GABA. Control refers to neurons derived from a healthy individual. [Figure 10B] Representative images are provided showing that the number of neurons was reduced in NEDAMSS patient cell lines compared to healthy controls after direct reprogramming from fibroblasts. Figure 10A provides staining using the pan-neuronal marker Tuj1, and Figure 10B provides staining for the neuron-specific marker GABA. Control refers to neurons derived from a healthy individual. [Figure 11] 1 provides a graph showing a comparison of neuronal conversion rate and neurite length, demonstrating reduced generation of neurons from fibroblasts in NEDAMSS patients. [Figure 12A]12A and 12B are graphs showing the expression of IRF2BPL mRNA (FIG. 12A) and IRF2BPL protein (FIG. 12B) between wild-type IRF2BPL levels and GFP levels when induced by five different promoters in HEK-293. [Figure 12B] 12A and 12B are graphs showing the expression of IRF2BPL mRNA (FIG. 12A) and IRF2BPL protein (FIG. 12B) between wild-type IRF2BPL levels and GFP levels when induced by five different promoters in HEK-293. [Figure 13] We present a strategy for designing three AAV gene therapy vectors encoding the wild-type IRE2BPL gene using truncated mutants of the p546 and endogenous pIRF promoters. [Figure 14-1] The annotated sequence of ssAAV9-p546+pIRFENH-5'UTR-IRF2BPL (SEQ ID NO: 3) is provided, showing the locations of the construction elements. [Figure 14-2] The annotated sequence of ssAAV9-p546+pIRFENH-5'UTR-IRF2BPL (SEQ ID NO: 3) is provided, showing the locations of the construction elements. [Figure 15-1] The annotated sequence of ssAAV9-pIRFSHORT-5'UTR-IRF2BPL (SEQ ID NO: 4) is provided, showing the locations of the construction elements. [Figure 15-2] The annotated sequence of ssAAV9-pIRFSHORT-5'UTR-IRF2BPL (SEQ ID NO: 4) is provided, showing the locations of the construction elements. [Figure 16-1] An annotated sequence of ssAAV9-p546-5'UTR-IRF2BPL (SEQ ID NO: 7) is provided, showing the locations of the construction elements. [Figure 16-2] An annotated sequence of ssAAV9-p546-5'UTR-IRF2BPL (SEQ ID NO: 7) is provided, showing the locations of the construction elements. DETAILED DESCRIPTION OF THE INVENTION

[0017] The interferon regulatory factor 2-binding protein-like (IRF2BPL) gene encodes a member of the IRF2BP family of transcriptional regulators.

[0018] IRF2BPL is an intronless gene located at 14q24.23. Transcripts are expressed in many organs, including components of the central nervous system (CNS), such as the cerebellum.

[0019] IRF2BPL mutations The wild-type cDNA sequence of IRF2BPL is set forth in SEQ ID NO:1. The nucleic acid sequence of the full-length IRF2BPL gene is set forth in SEQ ID NO:2. While the in vivo function of IRF2BPL in all species remains largely undefined, previous studies have demonstrated its role in the reproductive cycle of rodents and monkeys. Mutations in this gene have recently been associated with the childhood disorder NEDAMSS (neurodevelopmental disorder with regression, abnormal movements, speech loss, and seizures). NEDAMSS is inherited in an autosomal dominant manner. Little is known about the mechanism of this rare disease. A total of 18 patients worldwide are known to have this disorder.

[0020] In some embodiments, the IRF2BPL gene in the cells of the affected subject comprises one of the following mutations in SEQ ID NO: 1: Q126X, Q127X, E172X, Y173X, R188X, G195V, P372R, K418N, and / or a frameshift mutation at A708 (A708fs).

[0021] AAV gene therapy The present disclosure provides gene therapy vectors, e.g., rAAV vectors, that express the IRF2BPL gene and methods for treating NEDAMSS (neurodevelopmental disorders associated with regression, abnormal movements, speech loss, and seizures).

[0022] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 serotypes of AAV, which are characterized in general information and overviews of AAV, which can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and by heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0023] As used herein, "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0024] An "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed within the capsid. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, because such a vector is contained within an AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0025] Adeno-associated virus (AAV) is a replication-deficient parvovirus, whose single-stranded DNA genome is approximately 4.7 kb long, including a 145-nucleotide inverted terminal repeat (ITR). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is presented in GenBank Accession No. NC_002077, the complete genome of AAV-3 is presented in GenBank Accession No. NC_1829, the complete genome of AAV-4 is presented in GenBank Accession No. NC_001829, the AAV-5 genome is presented in GenBank Accession No. AF085716, the complete genome of AAV-6 is presented in GenBank Accession No. NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are presented in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is described in Gao et al. al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is presented in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is presented in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45 (2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in AAV2), the two rep promoters (p5 and p19) drive the generation of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0026] AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, enabling the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0027] Several studies have demonstrated long-term (more than 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscles are highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutics.

[0028] The recombinant AAV genome of the present disclosure comprises the nucleic acid molecule of the present disclosure and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and their derivatives). The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0029] The provided recombinant AAV (i.e., infectious, encapsidated rAAV particles) comprise an rAAV genome. The term "rAAV genome" refers to a polynucleotide sequence derived from a native AAV genome that has been modified. In some embodiments, the rAAV genome has been modified to remove the native cap and rep genes. In some embodiments, the rAAV genome comprises endogenous 5' and 3' inverted terminal repeats (ITRs). In some embodiments, the rAAV genome comprises ITRs from an AAV serotype different from the AAV serotype from which the AAV genome is derived. In some embodiments, the rAAV genome comprises a transgene of interest flanked at the 5' and 3' ends by inverted terminal repeats (ITRs). In some embodiments, the genome of an rAAV comprises a "gene cassette." In exemplary embodiments, both rAAV genomes lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome.

[0030] In some embodiments, the rAAV genome provided herein comprises one or more AAV ITRs flanking the transgene polynucleotide sequence. The transgene polynucleotide sequence is operably linked to transcriptional control elements (including, but not limited to, promoters, enhancers, and / or polyadenylation signal sequences) that are functional in target cells to form a gene cassette. Examples of promoters include the pIRF promoter (SEQ ID NO: 10), the chicken β-actin promoter (CBA) comprising the polynucleotide sequence set forth in SEQ ID NO: 6, and the P546 promoter comprising the polynucleotide sequence set forth in SEQ ID NO: 5. Additional promoters are contemplated herein, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters (e.g., but not limited to, actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, creatine kinase promoter, etc.).

[0031] Additionally, provided herein are pIRF promoter sequences, CB promoter sequences, P546 promoter sequences, and promoter sequences that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a pIRF (SEQ ID NO: 10), CBA (SEQ ID NO: 6), or P546 (SEQ ID NO: 5) sequence that exhibit transactivation activity.

[0032] Other examples of transcriptional control elements are tissue-specific control elements, such as promoters that allow specific expression in neurons or astrocytes. Examples include the neuron-specific enolase and glial fibrillary acidic protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline-regulated promoter. The gene cassette may also include an intron sequence to facilitate processing of the transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron.

[0033] The rAAV genomes provided herein comprise a polynucleotide encoding IRF2BPL (SEQ ID NO: 1). In some embodiments, the rAAV genomes provided herein comprise a polynucleotide sequence encoding a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by IRF2BPL cDNA (SEQ ID NO: 1).

[0034] Provided herein is a rAAV genome, in some embodiments, a polynucleotide sequence that encodes an IRF2BPL protein and hybridizes under stringent conditions to the polynucleotide sequence set forth in SEQ ID NO: 1 or its complement.

[0035] In exemplary embodiments, the rAAV genome comprises nucleotides 342-5299 of SEQ ID NO:3, or nucleotides 342-5031 of SEQ ID NO:4, or nucleotides 342-5070 of SEQ ID NO:7. In some embodiments, the rAAV genome provided herein comprises a polynucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 342-5299 of SEQ ID NO:3, or nucleotides 342-5031 of SEQ ID NO:4, or nucleotides 342-5070 of SEQ ID NO:7. In other embodiments, the rAAV genome provided herein comprises a polynucleotide sequence that hybridizes under stringent conditions to nucleotides 342-5299 of SEQ ID NO:3, or nucleotides 342-5031 of SEQ ID NO:4, or nucleotides 342-5070 of SEQ ID NO:7.

[0036] The DNA plasmid of the present disclosure contains the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAVrep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, or may be derived from an AAV serotype different from the rAAV genomic ITRs, including, but not limited to, AAV-9, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0037] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAVrep and cap genes, the AAVrep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0038] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Ratschin et al., Mol. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, and corresponding U.S. Pat. Nos. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine 13:1244-1250 (1995), Paul et al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis placed on the portions of the documents relating to rAAV production.

[0039] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 cells). In another embodiment, the packaging cells are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0040] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0041] The compositions provided herein comprise rAAV and one or more pharmaceutically acceptable excipients. Acceptable excipients are non-toxic to recipients and preferably inert at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate (e.g., phosphate-buffered saline (PBS)), citrate, or other organic acids; antioxidants such as ascorbic acid; low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as copolymers such as Tween, poloxamer 188, Pluronic® (e.g., Pluronic F68), or polyethylene glycol (PEG). The compositions provided herein can include a pharmaceutically acceptable aqueous excipient containing a non-ionic hypo-osmolar compound, such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, which can have one or more of the following properties: an osmolality of about 180 mgI / mL, about 322 mOsm / kg water vapor pressure osmometry, an osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20° C. and about 1.5 cp at 37° C., and a specific gravity of about 1.164 at 37° C. Exemplary compositions include about 20-40% non-ionic hypo-osmolar compound or contrast agent, or about 25% to about 35% non-ionic hypo-osmolar compound. An exemplary composition includes scAAV or rAAV viral particles formulated in 20 mM Tris (pH 8.0), 1 mM MgCl, 200 mM NaCl, 0.001% poloxamer 188, and about 25% to about 35% of a non-ionic, hypoosmotic compound. Another exemplary composition includes scAAV formulated in 1× PBS and 0.001% Pluronic F68.

[0042] The dosage of rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the time of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. Dosages may be expressed in units of viral genomes (vg). Dosages contemplated herein are 1×10 7 , 1×10 8 , 1×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 1×10 11 , about 1×10 12 , about 1×10 13 , about 1.1×10 13 , about 1.2×10 13 , about 1.3×10 13 , about 1.5×10 13 , about 2×10 13 , about 2.5×10 13 , about 3×10 13 , about 3.5×10 13 , about 4×10 13 , about 4.5×10 13 , about 5×10 13 , about 6×10 13 , about 1×10 14 , about 2×10 14 , about 3×10 14 , about 4×10 14 , about 5×10 14 , about 1×10 15 , about 1×10 16 Contains up to or more than 1 x 10 total viral genomes. 9 ~Approx. 1×10 10 vg, approx. 5×10 9 ~Approx. 5×10 10 vg, approx. 1×10 10 ~Approx. 1×10 11 vg, approx. 1×1011 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 15 vg, approx. 1×10 12 ~Approx. 1×10 14 vg, approx. 1×10 13 ~Approx. 6×10 14 vg, and approximately 6 × 10 13 ~Approx. 1.0×10 14 Dosages of 6×10 vg are also contemplated. 13 vg. Other doses exemplified herein are 1.5 x 10 13 vg.

[0043] Methods of transducing target cells with rAAV in vivo or in vitro are contemplated by the present disclosure. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present disclosure to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present disclosure is NEDAMSS.

[0044] Combination therapy is also contemplated by the present disclosure. As used herein, combination includes both simultaneous and sequential treatment. As a combination with a novel therapy, the combination of the method of the present disclosure with standard medical care is particularly contemplated.

[0045] Administration of an effective dose of the composition can be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intrathecal or other method of accessing cerebrospinal fluid, intraventricular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route of administration and serotype of the AAV components of the rAAV of the present disclosure (particularly the AAV ITRs and capsid proteins) can be selected and / or adapted by one skilled in the art taking into consideration the infection and / or disease state to be treated and the target cells / tissues expressing wild-type IRF2BPL protein.

[0046] The present disclosure provides for local administration and systemic administration of effective doses of rAAV and compositions of the present disclosure.For example, systemic administration refers to administration into the circulatory system so that the whole body is affected.Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration by injection, infusion or implantation.

[0047] The transduction of cells with the rAAV of the present disclosure results in the sustained expression of IRF2BPL protein.Therefore, the present disclosure provides a method for administering / delivering rAAV that expresses IRF2BPL protein to animals, preferably humans.These methods include transducing cells with one or more rAAV of the present disclosure.

[0048] The term "transduction" is used to refer to the administration / delivery of the coding region of IRF2BPL to recipient cells either in vivo or in vitro via a replication-deficient rAAV of the present disclosure, resulting in expression of IRF2BPL by the recipient cells.

[0049] The following examples are offered by way of illustration and not by way of limitation: The numerical ranges listed include each integer value within each range, including the stated integer minimum and maximum. [Example]

[0050] Example 1 - Characterization of iAstrocytes from NEDAMSS Patients Fibroblasts from patients with NEDAMSS (a neurodevelopmental disorder characterized by regression, abnormal movements, speech loss, and seizures) were converted into induced neural progenitor cells (iNPCs) as previously described (Meyer et al., PNAS 829-832 (2014)). Fibroblasts were obtained from four families harboring nonsense variants in the IRF2BPL gene that result in truncation of the RING finger domain. The IRF2BPL gene mutations represented are summarized below and in Figure 1. [Table 1] [Table 2]

[0051] Neural progenitor cells were cultured on fibronectin-coated dishes in NPC medium (DMEM / F12 medium containing 1% N2 supplement (Life Technologies), 1% B27, 1% antimicrobial (antibiotic-antimycotic) and 20 ng / ml fibroblast growth factor-2) until confluence. iAstrocytes were differentiated by seeding a small number of NPCs on separate fibronectin-coated dishes in astrocyte induction medium (DMEM medium containing 0.2% N2). These induced astrocytes are referred to herein as iAstrocytes or iAST. After 5 days of differentiation, induced astrocytes were seeded into either 96-well plates (10,000 cells / well), 384-well plates (2,500 cells / well), 24-well Seahorse plates (20,000 cells / well), or 96-well Seahorse plates (10,000 cells / well).

[0052] Immunohistochemistry was performed on primary fibroblasts from NEDAMSS patients and iastrocytes from the same patients using an antibody specific for IRF2BPL (Novus Biologics). As shown in Figure 2, IRF2BPL protein expression was not significantly different in primary fibroblasts from NEDAMSS patients (P1, P2, and P4), whereas cells from patient P3 showed reduced expression compared to fibroblasts from healthy individuals (H1, H2, H3, and H4). Similarly, as shown in Figure 3, IRF2BPL protein expression levels were similar in iastrocytes from NEDAMSS patients and healthy individuals, except for the cell line from patient P3, which showed significantly lower expression.

[0053] Figure 4A and B provide representative photographs of immunohistochemical staining of IRF2BPL (red staining) and DAPI (blue) in cell nuclei, and Figure 5 provides quantification of the abnormal cytoplasmic accumulation observed from the immunofluorescence images. The normalized ratio is the number of cells with cytoplasmic accumulation of IRF2BPL in astrocytes versus DAPI counts (n = 3). Blind hand counts were performed by two independent researchers. There were clear differences in the cytoplasmic IRF2BPL protein localization in the patient cell lines. These photographs and the graph provided in Figure 6 show that IRF2BPL was more highly accumulated in the cytoplasm of astrocytes derived from NEDAMSS patients (P1, P2, P3, and P4) than in the nuclei of astrocytes from healthy individuals (H1 and H3). Using the NPER extraction kit, we separated the two extracts and confirmed that the protein accumulated in the cytoplasm of the patient astrocytes.

[0054] Co-culture of iastrocytes with GFP-positive motor neurons derived from mouse stem cells (based on a publication by Meyer et al., PNAS 2014). Briefly, iastrocytes were plated in 96-well plates to form a monolayer. The following day, 10,000 FACS-sorted GFP-positive mouse motor neurons were added to each well. Neuronal survival and morphology were monitored for 3 days using an INCELL 6000 automated imager and analyzer software. Astrocytes from NEDAMSS patients were determined to be toxic or less supportive for motor neurons compared with iastrocytes from healthy controls. Figure 7A provides representative photographs of motor neurons in co-culture with iastrocytes from NEDAMSS patients and healthy individuals, and Figure 7B shows motor neuron viability. NEDAMSS astrocytes demonstrate significantly reduced motor neuron viability in co-culture compared with healthy astrocytes on day 3. Notably, only motor neurons are visible because they contain GFP (represented in black). In these photographs, motor neurons are visible due to GFP expression, but iastrocytes are not.

[0055] Figures 8 and 9 show that NEDAMSS patients have increased secretion of WNT1 compared to healthy patients. Dysregulation of the wnt signaling pathway may lead to neurodegeneration in NEDAMSS patients.

[0056] Example 2 - Neurons from NEDAMSS patients have reduced survival rates. Fibroblasts isolated from healthy individuals and NEDAMSS patients were differentiated into neurons as described in Hu et al., Cell Stem Cell, 17(2):204-12., 2015, incorporated herein by reference in its entirety. Fibroblasts were incubated with seven small molecules for seven days as described in Hu et al. (supra). This method does not use viruses expressing transcription factors. Figures 10A and 10B provide representative photographs showing that neurons derived from fibroblasts from NEDAMSS patients had reduced viability or differentiation potential. Figure 10A shows staining for the pan-neuronal marker Tuj1 in NEDAMSS patients (P1, P2, P3, and P4) and healthy individuals (H1 and H2). Figure 10B shows staining for the neuronal subtype marker Gabapentin in neurons from NEDAMSS patients (P1, P2, and P3) and healthy individuals (H2). Figure 11 provides quantification of the number of Tuj1+ neurons (Tuj1) and neurite length relative to DAPI from NEDAMSS patients (P1, P2, P3, and P4) and healthy individuals (H1 and H2) at day 7 of differentiation.

[0057] Example 3 - Constructs encoding IRF2BPL The human GFP cDNA clone was obtained from Origene, Rockville, MD. The GFP cDNA alone was engineered using: i) the 1034 base pair IRF2BPL promoter (pIRF Long ), ii) a 648 base pair fragment of the IRF promoter (pIRF Short ), iii) or a 296 base pair fragment of the IRF2BPL promoter (pIRF ENHThe constructs were further subcloned into the self-complementary AAV9 genome under the control of one or more of the following promoters: iv) the p546 promoter; or v) the hybrid chicken β-actin promoter (CB). The plasmid constructs also contained introns, such as the simian virus 40 (SV40) chimeric intron, 907 base pairs of the 5' untranslated region (UTR) of the full-length IRF2BPL gene, and the bovine growth hormone (BGH) polyadenylation signal (BGH PolyA). The constructs were packaged into the AAV9 genome. These constructs were generated to investigate the regulatory control and strength of the promoters. Briefly, the plasmids were transiently transfected into human embryonic kidney cells using calcium phosphate. After several days of incubation, GFP was monitored using a microscope to allow visualization, and expression was quantified by qPCR and Western blot. Figures 12A and 12B provide a comparison of IRF2BPL mRNA (Figure 12A) and IRF2BPL (Figure 12B) expression between wild-type IRF2BPL levels and GFP levels driven by five different promoters in HEK-293.

[0058] An exemplary polynucleotide construct encoding IRF2BPL was generated as described in Figure 13, and the AAV9 vector design used a full-length transcript of IRF2BPL under the control of a truncated mutant of the endogenous promoter. ENH The polynucleotide sequence of -5'UTR-IRF2BPL is the endogenous MECP2 promoter followed by the endogenous promoter pIRF 296 A truncated variant of ssAAV9-pIRF is set forth in the following SEQ ID NO: 3. SHORT The polynucleotide sequence of -5'UTR-IRF2BPL indicates that a truncated variant of the endogenous promoter is expressed in pIRF 648 The polynucleotide sequence of ssAAV9-p546-5'UTR-IRF2BPL is set forth in SEQ ID NO: 4, where the promoter is the endogenous MECP2 promoter. The annotated sequences showing the locations of the construction elements are shown in Figures 14, 15, and 16 and summarized in the table below. [Table 3] [Table 4] [Table 5]

[0059] References: Marcogliese et al., IRF2BPL Is Associated with Neurological Phenotypes. The American Journal of Human Genetics, 2018, 103(3). Meyer et al. Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. PNAS, 2014, 2014, 111(2):829-832 Hu et al. Direct Conversion of Normal and Alzheimer’s Disease Human Fibroblasts into Neuronal Cells by Small Molecules. Cell Stem Cell, 2015, 17(2):204-12. In particular embodiments, for example, the following items are provided: (Item 1) A polynucleotide comprising: (a) one or more regulatory control elements; (b) a polynucleotide comprising an interferon regulatory factor 2 binding protein-like (IRF2BPL) cDNA sequence. (Item 2) 2. The polynucleotide of item 1, wherein the regulatory control element is a pIRF promoter, a p546 promoter, or a CBA promoter, or a fragment thereof. (Item 3) <​​​ (Item 5) A recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequence according to any one of items 1 to 4. (Item 6) 6. The rAAV of item 5, wherein the genome comprises a pIRF promoter and an IRF2BPL cDNA. (Item 7) 6. The rAAV of item 5, wherein the genome comprises a p546 promoter and an IRF2BPL cDNA. (Item 8) 6. The rAAV of item 5, wherein the genome comprises a CBA promoter and an IRF2BPL cDNA. (Item 9) 6. The rAAV of item 5, wherein the genome comprises a pIRF promoter, a p546 promoter, and an IRF2BPL cDNA. (Item 10) 10. The rAAV of any one of items 5 to 9, wherein the rAAV is of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and derivatives thereof. (Item 11) An rAAV particle comprising the rAAV described in any one of items 5 to 10. (Item 12) A method for treating an interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder in a subject in need thereof, comprising administering the rAAV described in any one of items 5 to 10 or the rAAV particle described in item 11. (Item 13) 12. The method of item 11, wherein the disorder comprises NEDAMSS (neurodevelopmental disorder with regression, abnormal movements, speech loss, and seizures). (Item 14) Use of the rAAV of any one of items 5 to 10 or the rAAV particle of item 11 in the preparation of a medicament for the treatment of an interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder. (Item 15) A composition comprising the rAAV of any one of items 5 to 10 or the rAAV particle of item 11 for the treatment of an interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder.

Claims

1. A polynucleotide comprising: (a) one or more regulatory control elements, one of said regulatory control elements being a pIRF promoter, said pIRF promoter comprising SEQ ID NO: 10, SEQ ID NO: 9, or SEQ ID NO: 8 operably linked to a p546 promoter; (b) an interferon regulatory factor 2 binding protein-like (IRF2BPL) cDNA sequence.

2. The polynucleotide of claim 1 , wherein the p546 promoter comprises SEQ ID NO:

5.

3. 3. The polynucleotide of claim 1 or claim 2, wherein the IRF2BPL cDNA comprises the polynucleotide sequence set forth in SEQ ID NO:

1.

4. A recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequence of any one of claims 1 to 3.

5. A recombinant adeno-associated virus (rAAV), comprising: (a) one or more regulatory control elements, wherein one of said regulatory control elements is a pIRF promoter, or one of said regulatory control elements is a pIRF promoter operably linked to a p546 promoter; (b) an IRF2BPL cDNA sequence comprising the polynucleotide sequence of SEQ ID NO: 1; and rAAV having a genome comprising:

6. 6. The rAAV of claim 5, wherein the IRF2BPL cDNA sequence comprises the polynucleotide sequence of SEQ ID NO:

1.

7. 7. The rAAV of claim 5 or claim 6, wherein one of the regulatory control elements comprises SEQ ID NO: 10, SEQ ID NO: 9 or SEQ ID NO: 8 operably linked to the p546 promoter.

8. The rAAV of any one of claims 4 to 7, wherein the p546 promoter comprises SEQ ID NO:

5.

9. The rAAV according to any one of claims 4 to 8, wherein the rAAV is of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRH10, AAVRH74, AAV11, AAV12, AAV13, or Anc80, AAV7m8, and derivatives thereof.

10. An rAAV particle comprising the rAAV of any one of claims 4 to 9.

11. A composition comprising the rAAV of any one of claims 4 to 9 or the rAAV particle of claim 10 for the treatment of an interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder.

12. 12. The composition of claim 11, wherein the interferon regulatory factor 2-binding protein-like (IRF2BPL)-associated disorder comprises NEDAMSS (neurodevelopmental disorder with regression, abnormal movements, speech loss, and seizures).

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