Gene therapy for 21-hydroxylase deficiency

Abstract

Disclosed herein are recombinant adeno-associated viral vectors that express 21-hydroxylase (21OH) protein and related uses for treating 21OH deficiency.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 163,634, filed March 19, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically herewith are incorporated by reference in their entirety herein: Computer-readable copy of the sequence listing (Filename: ADRE_002_01WO_SL, Recorded date: March 17, 2022, File size: 28.5 kilobytes). [Technical field]

[0003] The present disclosure generally relates to the field of gene therapy. In particular, the present disclosure describes recombinant adeno-associated virus (rAAV) vectors and particles that express 21-hydroxylase (21OH) protein. The rAAV vectors and rAAV particles may be used to treat 21OH deficiency. [Background technology]

[0004] 21-hydroxylase (21OH) is a cytochrome P450 enzyme encoded by the CYP21A2 gene and involved in the biosynthesis of the steroid hormones aldosterone and cortisol, which are synthesized in the adrenal cortex. A high recombination rate between the functional CYP21A2 gene and the closely linked non-functional CYP21A1P pseudogene results in a relatively high incidence of congenital adrenal hyperplasia (CAH) and its abnormal genetic features, which are caused by gene conversion rather than point mutations. Defects in CYP21A2 cause 21-hydroxylase deficiency (21OHD), which leads to either i) CAH, with fetal virilization of the female external genitalia, reduced or absent production of glucocorticoids and mineralocorticoids, and massive androgen excess ("classical" 21OHD), or ii) a milder form of the disease in which there is increased androgen production but no virilization or clinically significant deficiency of cortisol or aldosterone ("non-classical" 21OHD).

[0005] After decades of treatment strategies, management of severe forms of 21OHD remains a clinical challenge. Although patients can be treated with exogenous steroids, infants and adult patients remain at risk for adrenal crisis (the failure of the adrenal glands to respond to physical stresses such as routine infection, trauma, or strenuous exertion). Adrenal crisis can rapidly lead to severe shock and death, even in well-educated and adherent patients. See Hahner et al., J. Clin. Endocrinol. Metab, Feb;100(2):407-416(2015). In addition, there are significant consequences related to growth, sex, and sexuality. In female patients, it is inherently difficult to suppress adrenal androgen production using supraphysiological doses of glucocorticoids. The result is alternating androgen excess and glucocorticoid excess that may lead to short stature, obesity, repeated childhood genital surgeries, pubertal changes, and chronic virilization. Hyperandrogenism remains a major cosmetic burden for female patients suffering from classical and non-classical forms of the disease due to hirsutism, male muscular development, enlargement of the clitoral size, and sexual dysfunction. See Gastaud et al., J. Clin. Endocrinol. Metab, 92(4), 1391-1396 (2007). Male patients are at risk for short stature and early virilization. In some patients, treatment failure may even lead to bilateral adrenalectomy (Gmyrek et al., Pediatrics, 109:E28 (2002); Bruining et al., J. Clin. Endocrinol. Metab, 86:482-484 (2001)). There remains a need for treatments that allow for durable correction of 21OHD. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Hahner et al.,J.Clin.Endocrinol.Metab,Feb;100(2):407-416(2015) [Non-Patent Document 2] Gastaud et al.,J.Clin.Endocrinol.Metab,92(4),1391-1396(2007) [Non-Patent Document 3] Gmyrek et al., Pediatrics, 109:E28(2002) [Non-Patent Document 4] Bruining et al., J. Clin. Endocrinol. Metab, 86:482-484 (2001) Summary of the Invention [Means for solving the problem]

[0007] The present disclosure provides methods and compositions for use in treating hormone imbalance, and in particular 21-hydroxylase (21OH) deficiency (21OHD), such as congenital adrenal hyperplasia (CAH). The methods provided herein include administration of a viral vector comprising a nucleic acid molecule encoding a 21OH protein.

[0008] In one aspect, the disclosure provides a method comprising administering to a subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; and wherein the therapeutically effective amount is about 10 per kilogram. 12 Vector genome (vg / kg) ~ approx. 10 14 vg / kg range.

[0009] In some embodiments, the subject requires expression of 21OH. In some embodiments, the subject has 21OH deficiency (21OHD). In some embodiments, the subject is female and has external genital anomalies according to the Prader classification. In some embodiments, the subject has congenital adrenal hyperplasia (CAH). In some embodiments, the CAH is classical CAH. In some embodiments, administration of the rAAV vector results in expression of 21OH in the subject. In some embodiments, 21OH is expressed in the adrenal cortex, adrenal medulla, adrenal stem cells, adrenal progenitor cells, liver, or ovaries of the subject.

[0010] In some embodiments, the subject is in need of cortisol and / or aldosterone. In some embodiments, the subject has a cortisol deficiency and / or an aldosterone deficiency. In some embodiments, the subject has an excess of progesterone, 17-OHP, renin, androstenedione (A4) and / or 11-ketosteroids. In some embodiments, the subject has an excess of progesterone, 17-OHP, renin, androstenedione (A4) and / or 11-ketosteroids in the blood and / or urine.

[0011] In another aspect, the disclosure provides a method for expressing 21-hydroxylase (21OH) in a subject in need of expression of 21-hydroxylase (21OH). In some embodiments, such a method comprises administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); and (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter, and the therapeutically effective amount is about 10 12 vg / kg~about 10 14 vg / kg range, thereby expressing 21OH in the subject.

[0012] In some embodiments, 21OH is expressed in the adrenal cortex, adrenal medulla, adrenal stem cells, adrenal progenitor cells, liver, or ovaries of the subject. [Brief description of the drawings]

[0013] [Figure 1] A shows increased weight gain over time in mice treated with the indicated doses of AAV5 encoding the 21-hydroxylase (21OH) protein compared to untreated control mice. B shows a dose-dependent increase in AAV5 viral genome encoding the 21-hydroxylase (21OH) protein detected in the adrenal glands of a mouse model (triangles) or non-human primate (NHP, squares). C shows the amount of human 21-OH produced, expressed as a percentage of endogenous 21-OH in the adrenal glands of a mouse model (triangles) or non-human primate (NHP). [Diagram 2] A timeline of a Phase 1 / 2 first-in-human, open-label, dose-escalation study in adults with classic CAH due to 21-OHD is shown. [Diagram 3] A timeline of possible dose escalation and dose expansion in Phase 1 / 2 clinical trials is shown. [Figure 4] The design of the AAV-hCYP21-Opt vector, the active component of BBP-631, is shown, which contains the following elements from 5' to 3': AAV5'ITR, AAV packaging signal (Ψ), cytomegalovirus enhancer, chicken β-actin promoter containing a canonical Kozak sequence with a splice donor and a rabbit β-globin intron with a splice acceptor, codon-optimized cDNA of human 21-hydroxylase (21-OH), rabbit β-globin polyA signal, and AAV3'ITR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present disclosure relates to recombinant adeno-associated virus (AAV) vectors that can be engineered to express 21-hydroxylase (21OH) and used to treat 21-hydroxylase deficiency (21OHD), including congenital adrenal hyperplasia (CAH). In some aspects, the disclosure provides methods of treatment, expression of 21OH, and / or hormone modulation, and compositions for use therein, that include administration of a recombinant AAV vector that includes a non-AAV nucleotide sequence encoding a 21-OH protein.

[0015] The current approach to the clinical management of CAH is to address primary adrenal insufficiency and treat with exogenous glucocorticoids (and mineralocorticoids when necessary) to normalize adrenal androgens, which often requires supraphysiological doses of glucocorticoids. Because adequate control of adrenal androgen levels often requires supraphysiological doses of glucocorticoids, lifelong glucocorticoid replacement in CAH is associated with significant morbidity, including shortened overall life span, cardiovascular disease, metabolic disease, bone disease, and short stature. In addition, classic CAH patients with a salt-wasting phenotype also require lifelong mineralocorticoid replacement.

[0016] In addition to androstenedione (A4), other adrenal hormones, especially 17-OHP, are often used to guide management as well. Although the clinical management paradigm may seem straightforward, clinicians face a very difficult balancing act when adjusting maintenance glucocorticoid dosing in both children and adults with CAH. For example, clinicians attempt to treat children with the lowest and safest maintenance doses of glucocorticoids possible, especially to preserve growth potential and final adult height. Although supraphysiologic high-dose glucocorticoid replacement therapy may help protect both pediatric and adult patients from symptoms associated with hyperandrogenism, it also carries the risk of numerous long-term metabolic, skeletal, and cardiovascular sequelae. Because CAH patients receive exogenous glucocorticoids and / or mineralocorticoids from infancy, current treatment approaches impose a high burden of care on patients and caregivers, leading to poor adherence to current standards of care.

[0017] There is substantial morbidity associated with the disease and current standard of care, including shortened overall life span, cardiovascular, metabolic, and bone disease, and short stature. In addition, adrenal crisis can rapidly lead to severe shock and death, necessitating adequate patient and caregiver education regarding stress dosing of glucocorticoids. Classical CAH patients receiving current standard of care remain burdened with lifelong disease, including a fivefold higher all-cause mortality rate. Therefore, alternative therapies that could avoid the need for long-term supraphysiologic glucocorticoids and restore endogenous cortisol production would be highly desirable for all patients with classic CAH.

[0018] The present disclosure provides recombinant adeno-associated virus (AAV) vectors that are engineered to express 21-hydroxylase (21OH) and can be used to treat 21-hydroxylase deficiency (21OHD). In some aspects, the present disclosure provides recombinant adeno-associated virus (rAAV) vectors that include a non-AAV nucleotide sequence encoding a 21OH protein, rAAV particles that include such vectors, and methods of using such vectors and particles to treat 21OHD in a subject in need thereof.

[0019] In some embodiments, the compositions and methods disclosed herein restore adrenal cortical cell function by providing endogenous and physiological pathways for the synthesis of glucocorticoids and mineralocorticoids. Thus, in some embodiments, the compositions and methods disclosed herein reduce the reliance on exogenous glucocorticoids (e.g., hydrocortisone) and / or mineralocorticoids for disease management, thereby significantly reducing the deleterious effects of either excess glucocorticoid replacement or excess levels of serum androgens. In some embodiments, the compositions and methods disclosed herein alleviate at least one symptom or clinical feature of CAH associated with high levels of precursors such as 17-OHP and androgens such as androstenedione (A4) and 11-oxo or 11-keto sterols (e.g., 11-keto-testosterone). Thus, in some embodiments, the compositions and methods disclosed herein reduce the hyperandrogenism associated with 21-OHD. In some embodiments, the compositions and methods disclosed herein reduce the risk of life-threatening adrenal crisis.

[0020] The headings used herein are merely for organizational purposes and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated documents or portions of documents defines a term and it conflicts with the definition of that term in this application, the definition set forth in this application shall control. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, any form of acknowledgment or suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

[0021] Any concentration range, percentage range, ratio range, or integer range herein should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless otherwise specified. The term "about," when immediately preceding a number or numeral, means that the number or numeral is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the indicated components, unless otherwise specified. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives following. As used herein, the terms "include" and "comprise" are used interchangeably.

[0022] Recombinant AAV vectors and particles In one aspect, the disclosure provides a viral vector for delivering a nucleic acid sequence of 21-hydroxylase (21OH) to a cell in need of treatment. Thus, in one embodiment, the disclosure relates to a recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence (also referred to as a heterologous polynucleotide) encoding a 21OH protein, the non-AAV nucleotide sequence being operably linked to a promoter. As used herein, the term "operably linked" or "operably linked" refers to the physical or functional juxtaposition of the described components that allows the components to function in the intended manner. In the example of an expression control element (e.g., a promoter or enhancer) operably linked to a polynucleotide, the relationship is such that the control element regulates the expression of the nucleic acid. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (in cis or trans) in such a relationship that at least one DNA sequence can exert a physiological effect on the other sequence. "Operably linked" may mean that the nucleic acid sequences being linked are contiguous or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame.

[0023] In some embodiments, the rAAV vector expresses a 21OH protein that is a human 21OH protein. The CYP21A2 gene encodes a 21OH protein. As used herein, 21OH may refer to a 21OH protein or a nucleic acid sequence encoding the protein. In some cases, the 21OH protein expressed by the rAAV vector described herein is a native (e.g., wild-type) 21OH protein. The 21OH protein or 21OH polypeptide encoded by the nucleotide sequence includes the full-length native sequence, as in the naturally occurring 21OH protein, as well as functional subsequences, modifications, or sequence variants, so long as the subsequences, modifications, or variants retain some functionality of the native full-length 21OH protein. In the methods and uses of the present disclosure, the 21OH protein and 21OH polypeptide encoded by the nucleotide sequence in the rAAV vector can be, but need not be, identical to the endogenous 21OH protein that is missing or insufficiently expressed or deficient in the subject to be treated.

[0024] In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding a 21OH protein is a wild-type CYP21 gene sequence. In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding a 21OH protein is codon-optimized with respect to the wild-type CYP21 gene sequence. In some embodiments, the nucleotide sequence encoding a 21OH of the present disclosure is a codon-optimized sequence and comprises or consists of SEQ ID NO:50 (see Table 1). In some embodiments, the nucleotide sequence encoding a 21OH is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:50.

[0025] Codon optimization takes advantage of the redundancy of the genetic code, allowing nucleotide sequences to be altered while maintaining the same amino acid sequence of the encoded protein. In some embodiments, codon optimization is performed to facilitate increased or decreased expression of the encoded protein. This is accomplished by tailoring the codon usage in a nucleotide sequence to a particular cell type, thereby taking advantage of the cellular codon bias that corresponds to the bias in the relative abundance of a particular tRNA in the cell type. By altering the codons in the nucleotide sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Conversely, expression can be decreased by selecting codons whose corresponding tRNAs are known to be rare in a particular cell type.

[0026] In some embodiments, the codon-optimized nucleotide sequences encoding 21OH proteins are more stable than the wild-type cDNA sequences, thereby avoiding the generation of alternatively spliced ​​variants or truncated proteins when non-AAV nucleotide sequences are introduced into the transcription machinery via gene therapy.

[0027] In some embodiments, the non-AAV nucleotide sequence (e.g., a heterologous sequence) encoding a 21OH protein encodes the amino acid sequence of SEQ ID NO: 1 (see Table 1). In other embodiments, the non-AAV nucleotide sequence encoding a 21OH protein encodes an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0028] [Table 1-1] [Table 1-2] [Table 1-3]

[0029] In some embodiments, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding 21OH protein is a cDNA of human 21OH, optionally linked to a nucleotide sequence encoding a hemagglutinin (HA) tag. In certain cases, the non-AAV nucleotide sequence (e.g., heterologous sequence) encoding 21OH protein is linked to a nucleotide sequence encoding a tag, such as hemagglutinin (HA), UA, cMyc, or any suitable tag. "CYPHA" may refer to a 21OH transgene fused to a sequence encoding a HA tag.

[0030] The terms "identity", "homology" and their grammatical variations mean that two or more referenced entities are the same when they are "aligned" sequences. Thus, as an example, if two polypeptide sequences are identical, they have the same amino acid sequence at least within the referenced region or portion. If two polynucleotide sequences are identical, they have the same polynucleotide sequence at least within the referenced region or portion. Identity can span a defined area (region or domain) of a sequence. An "area" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, if two protein or nucleic acid sequences are identical over one or more sequence areas or sequence regions, they share identity within that region. An "aligned" sequence refers to a sequence of multiple polynucleotides or proteins (amino acids), often containing corrections of missing or additional bases or amino acids (gaps) compared to a reference sequence.

[0031] Identity can span the entire length of a sequence or a portion of a sequence. In certain embodiments, the length of sequences that share percent identity is 2, 3, 4, 5 or more consecutive polynucleotides or amino acids, such as at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids. In further certain embodiments, the length of sequences that share identity is 20 or more consecutive polynucleotides or amino acids, such as at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive amino acids. In further specific embodiments, the length of sequences sharing identity is 35 or more consecutive polynucleotides or amino acids, e.g., at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more consecutive amino acids. In yet further specific embodiments, the length of sequences sharing identity is 50 or more consecutive polynucleotides or amino acids, e.g., at least 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-110, or more consecutive polynucleotides or amino acids.

[0032] The term "homologous" or "homology" means that two or more referenced entities share at least partial identity over a given region or portion. An "area," "region," or "domain" of homology or identity means that a portion of two or more referenced entities shares homology or is the same. Thus, if two sequences are identical over one or more sequence regions, they share identity in these regions. "Substantial homology" means that the molecule is structurally or functionally conserved such that it has, or is predicted to have, at least a partial structure or function (e.g., biological function or activity) of the reference molecule, or a related / corresponding region or portion of the reference molecule with which it shares homology.

[0033] The degree of identity (homology) between two sequences can be ascertained using computer programs and mathematical algorithms. Percent identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk / Tools / msa / clustalo using default parameters. See Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.” (2011, October, 11), Molecular systems biology, 7:539. For the purposes of calculating identity to sequences, extensions such as tags are not included.

[0034] Vector genome sequences, including the rAAV vector genome sequences described herein, can include one or more "expression control elements." Typically, an expression control element is a nucleic acid sequence that affects the expression of an operably linked polynucleotide. Control elements, including the expression control elements shown herein, such as promoters and enhancers, present in the vector are included to facilitate transcription and / or translation (e.g., promoters, enhancers, splicing signals of introns, maintaining the correct reading frame of the gene to allow in-frame translation of mRNA, etc.) of the appropriate heterologous polynucleotide (e.g., 21OH gene). Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, in some cases, sequences that enhance secretion of the encoded product (e.g., 21OH). In some embodiments, the rAAV vector genome sequence of the disclosure comprises a consensus sequence such as a Kozak sequence (e.g., a DNA sequence that is transcribed into an RNA Kozak sequence). In some embodiments, the rAAV vector genome sequence of the disclosure comprises a Kozak sequence upstream of a nucleotide sequence encoding a 21OH protein. In some embodiments, the RNA Kozak sequence comprises or consists of ACCAUGG (SEQ ID NO: 44), GCCGCCACCAUGG (SEQ ID NO: 45), CCACCAUG (SEQ ID NO: 46), or CCACCAUGG (SEQ ID NO: 47).

[0035] Expression control can occur at the level of transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end of the transcribed polynucleotide (i.e., "upstream"). Expression control elements can also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., within an intron). Expression control elements can be located far from the transcribed sequence, even far away (e.g., 100-500, 500-1000, 2000-5000, 5000-10,000 nucleotides, or more nucleotides from the nucleotide sequence expressing the 21OH). Nevertheless, in the case of AAV vectors, due to polynucleotide length limitations, such expression control elements will usually be within 1-1000 nucleotides from the nucleotide sequence encoding the 21OH.

[0036] Functionally, the expression of an operably linked nucleotide sequence encoding a 21OH can be at least partially controlled by an element (e.g., a promoter) such that the element regulates the transcription of the nucleotide sequence and, optionally, the translation of the transcript. A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' of the transcribed sequence, 3' of the transcribed sequence, or within the transcribed sequence.

[0037] As used herein, a "promoter" can refer to a nucleic acid sequence that is located adjacent to a nucleic acid sequence (e.g., a heterologous polynucleotide) that encodes a recombinant product (e.g., a 21OH). A promoter is usually operably linked to an adjacent sequence, e.g., a heterologous polynucleotide. A promoter usually increases the amount of expression from a heterologous polynucleotide compared to the amount expressed in the absence of the promoter.

[0038] As used herein, "enhancer" can refer to a sequence located adjacent to a nucleotide sequence encoding 21OH. Enhancer elements are usually located upstream of promoter elements, but can also function and be located downstream of or within a DNA sequence (e.g., a nucleotide sequence encoding 21OH). Thus, enhancer elements can be located, for example, 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a heterologous polynucleotide. Enhancer elements usually increase the expression of a heterologous polynucleotide beyond the level of increased expression provided by a promoter element.

[0039] In some embodiments, expression control elements include ubiquitous, constitutive, or promiscuous promoters and / or enhancers capable of driving expression of the polynucleotide in many different cell types. Such elements include the cytomegalovirus / β-actin hybrid (e.g., CAG, CB6, or CBA) promoter, the phosphoglycerol kinase (PGK) promoter, the cytomegalovirus (CMV) immediate early promoter and / or enhancer sequence, the Rous sarcoma virus (RSV) promoter and / or enhancer sequence, as well as other viral promoters and / or enhancers that are active in a variety of mammalian cell types, or synthetic elements that do not occur in nature (e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the chicken β-actin (CBA) promoter, the EF1 promoter (Invitrogen), the immediate early CMV enhancer in combination with the CBA promoter (Beltran et al., Gene Therapy, 17(9):1162-1174 (2010)), and the CBh promoter (Gray et al., Hum Gene Ther, 22(9):1143-1153 (2011)). In certain aspects, the rAAV of the present disclosure comprises a synthetic CASI promoter that includes a portion of the CMV enhancer, a portion of the chicken beta actin promoter, and a portion of the UBC enhancer. See, e.g., WO2012 / 115980. In some embodiments, the rAAV vector comprises a CAG promoter sequence that includes SEQ ID NO:2 or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2.In some embodiments, the rAAV vector comprises a PGK promoter sequence comprising SEQ ID NO:3 or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3. In some embodiments, the rAAV vector comprises a CB6 promoter sequence comprising SEQ ID NO:48 or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:48. In some embodiments, the rAAV vector comprises a CBA promoter sequence comprising SEQ ID NO:49 or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:49. See Table 1 for non-limiting examples of promoter sequences.

[0040] Inducible promoters allow the control of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, specific physiological conditions, acute phase, specific differentiation states of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen and Clontech. Many other systems have been described and can be used. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline repression system, the tetracycline induction system, the RU486 induction system, and the rapamycin induction system. Any type of inducible promoter may be used that is tightly regulated and specific to the particular target cell type in which 21OH expression is intended.

[0041] Expression control elements (e.g., promoters) include those that are active in a particular tissue or cell type, referred to herein as "tissue-specific expression control elements / promoters." Tissue-specific expression control elements are typically active in a particular cell or tissue (e.g., cells of the adrenal gland, adrenal cortex, liver, brain, central nervous system, spinal cord, eye, retina, bone, muscle, lung, pancreas, heart, kidney, etc.). Expression control elements are typically active in a particular cell, tissue, or organ type because they are recognized by a transcription activator protein or other transcription regulator specific to those cells, tissues, or organs. Thus, in some cases, the rAAV vectors of the present disclosure include a promoter that directs expression of a nucleotide sequence encoding a 21OH protein in a host cell (e.g., an adrenal cell). In certain embodiments, the adrenal cell is an adrenal cortex cell. In some embodiments, the rAAV vectors of the present disclosure include a non-AAV nucleotide sequence encoding a 21OH protein, which is operably linked to a promoter specific for expression in an adrenal cortex cell or an adrenal medulla cell. In some embodiments, the rAAV vector of the present disclosure comprises a non-AAV nucleotide sequence encoding a 21OH protein, the non-AAV nucleotide sequence being operably linked to a promoter specific for expression in the adrenal gland (e.g., adrenal cortex or adrenal medulla), liver or ovary of a subject. In some embodiments, the rAAV vector of the present disclosure comprises a non-AAV nucleotide sequence encoding a 21OH protein, the non-AAV nucleotide sequence being operably linked to a promoter specific for expression in an adrenal stem cell (e.g., adrenal cortex stem cell) or adrenal progenitor cell.

[0042] In some embodiments, the regulatory sequence useful in the rAAV vector of the present disclosure also contains an intron, which is optionally located between the promoter / enhancer sequence and the 21OH gene. In some embodiments, the intron sequence is a 100 bp mini-intron splice donor / splice acceptor derived from SV-40, called SD-SA. In some embodiments, the rAAV vector comprises a post-transcriptional regulatory element. One example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional element (WPRE). (See, e.g., Wang and Verma, Proc. Natl. Acad. Sci., USA, 96:3906-3910 (1999)). In certain embodiments, the post-transcriptional regulatory element is the hepatitis B virus post-transcriptional regulatory element (HBVPRE) or the RNA transport element (RTE). In some embodiments, the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in U.S. Patent No. 6,136,597 or U.S. Patent No. 6,287,814. In some embodiments, the WPRE sequence comprises or consists of: aatcaacctc tggattacaa aatttgtgaa agattgactg atattcttaa ctatgttgct ccttttacgc tgtgtggata tgctgcttta atgcctctgt atcatgctat tgcttcccgt acggctttcg ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tccgtcaacg tggcgtggtg tgctctgtgt ttgctgacgc aacccccact ggctggggca ttgccaccac ctgtcaactc ctttctggga ctttcgcttt ccccctcccg atcgccacgg cagaactcat cgccgcctgc cttgcccgct gctggacagg ggctaggttg ctgggcactg ataattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccaactggat cctgcgcggg acgtccttct gctacgtccc ttcggctctc aatccagcgg acctcccttc ccgaggcctt ctgccggttc tgcggcctct cccgcgtctt cgctttcggc ctccgacgag tcggatctcc ctttgggccg cctccccgcc tg (sequence number 51).

[0043] In some embodiments, the rAAV vector comprises a polyA signal, which may be derived from a number of suitable species, including but not limited to SV-40, human, and bovine.

[0044] Another useful regulatory component that may be included in a rAAV vector is an internal ribosome entry site (IRES). An IRES sequence or other suitable system may be used to generate more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to generate a protein containing more than one polypeptide chain, or to express two different proteins from or within the same cell. An exemplary IRES is the poliovirus internal ribosome entry sequence. An IRES may be located 5' or 3' of the 21OH transgene in a rAAV vector. In other embodiments, a rAAV vector may include a nucleotide sequence encoding a 2A peptide, which allows for the expression of multiple polypeptides from a single promoter.

[0045] A recombinant "vector" or "rAAV vector" is derived from the wild-type genome of a virus such as AAV by using molecular methods to remove the wild-type genome from the virus and replace it with a non-native nucleic acid, such as a heterologous polynucleotide sequence (e.g., a therapeutic gene expression cassette expressing 21OH). Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the AAV vector. A rAAV vector can be distinguished from a viral genome because all (or a portion) of the viral genome has been replaced with a non-native sequence with respect to the viral genome nucleic acid. Thus, the incorporation of a non-native sequence, such as a heterologous polynucleotide, defines the viral vector as a "recombinant" vector, and in the case of AAV, can be referred to as a "rAAV vector." A rAAV vector containing a nucleic acid molecule encoding 21OH may also be referred to as a "CYP21 vector" or a "21OH vector." As the context makes clear, a "vector" may refer to an isolated recombinant nucleotide sequence, or an AAV particle or AAV virion containing a recombinant nucleotide sequence.

[0046] In some embodiments, the rAAV vector does not contain any binding site for miRNA (microRNA). In some embodiments, the rAAV vector contains one, two, three, four, five or more binding sites for miRNAs expressed in cells where expression of 21OH proteins is undesirable (i.e., detargeted). In some embodiments, the rAAV vector contains one or more binding sites for miR-122. Binding of miR-122 to a sequence encoding 21OH may reduce expression of this sequence in liver cells where miR-122 is highly prevalent (Thakral and Ghoshal, Curr. Gene Ther. 2015;15(2):142-150).

[0047] The rAAV nucleic acid sequences can be packaged into viruses (also referred to herein as "particles" or "virions") for subsequent infection (transformation) of cells in vitro, in vitro, or in vivo. When recombinant vector sequences are encapsidated or packaged into an AAV particle, the particle can be referred to as "rAAV." Such particles or virions typically include proteins that encapsidate or package the vector genome. Particular examples include viral envelope proteins and, in the case of AAV, capsid proteins.

[0048] The AAV components of the rAAV vectors and rAAV particles described herein may be selected from various AAV serotypes. In certain cases, the rAAV vector may contain AAV nucleic acid sequences derived from rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh74 serotypes. These AAV components may be readily isolated from AAV serotypes using a variety of techniques. Such AAV may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be obtained via synthetic or other suitable means by reference to published sequences available in the literature or databases such as, for example, GenBank™, PubMed, and the like.

[0049] In certain embodiments, rAAV vectors or particles comprise AAV nucleic acid sequences or AAV proteins, for example, as disclosed in U.S. Patent No. 7,906,111 or U.S. Patent No. 7,629,322, the entirety of which is incorporated herein by reference. In some embodiments, rAAV vectors or particles comprise AAV nucleic acid sequences or AAV proteins from AAV serotype AAV8 or its variants, for example, as disclosed in U.S. Patent No. 7,282,199, U.S. Patent No. 9,587,250 or U.S. Patent No. 9,677,089, the entirety of which is incorporated herein by reference. In some embodiments, rAAV vectors or particles comprise AAV nucleic acid sequences or AAV proteins from AAV serotype AAV9 or its variants, for example, as disclosed in U.S. Patent No. 7,198,951, the entirety of which is incorporated herein by reference. In some embodiments, the rAAV vector or rAAV particle comprises AAV nucleic acid sequences or AAV proteins derived from AAV serotype rh74 or a variant thereof, for example, as disclosed in U.S. Pat. No. 9,840,719, the entire contents of which are incorporated by reference herein.

[0050] In some aspects, the rAAV vector of the present disclosure comprises a nucleic acid molecule comprising at least one AAV ITR sequence. In certain embodiments, the rAAV vector comprises two ITR sequences, which may be of the same or different AAV serotypes. In certain cases, the AAV ITRs may be selected from any AAV serotype, including, but not limited to, rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh74, and other AAV serotypes. In some embodiments, the rAAV vector described herein comprises a genome comprising one or two AAV2 ITR sequences.

[0051] The present disclosure further provides rAAV particles comprising the rAAV vectors described herein.Thus, in some aspects, the present disclosure relates to rAAV particles comprising a nucleic acid molecule comprising at least one AAV ITR and a non-AAV nucleotide sequence (also referred to as heterologous polynucleotide) encoding a 21OH protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter.In some embodiments, the rAAV particles comprise at least one capsid protein selected from the group consisting of AAV serotypes rh10, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh74, and other AAV serotypes.

[0052] In one embodiment, the rAAV vector is a rh10 AAV vector comprising a cDNA of human 21OH, ITRs from AAV2, and a CAG promoter consisting of an enhancer, promoter, splice donor and intron from chicken β-actin gene, and a splice acceptor from rabbit β-globin gene from cytomegalovirus. In a further embodiment, the rAAV vector comprises a nucleic acid sequence encoding a 21OH protein comprising SEQ ID NO:1 and a CAG promoter comprising or consisting of SEQ ID NO:2. In one embodiment, the rAAV vector is a rh10 AAV vector comprising a cDNA of human 21OH, ITRs from AAV2, and a PGK promoter. In another embodiment, the rAAV vector comprises a nucleic acid sequence encoding a 21OH protein comprising SEQ ID NO:1 and a PGK promoter comprising or consisting of SEQ ID NO:3.

[0053] In some embodiments, the rAAV vector or particle comprises an AAV1 capsid and a nucleic acid molecule comprising a human 21OH cDNA; a CAG, PGK, CBA or CB6 promoter; and optionally one or two AAV2 ITR sequences. In one embodiment, the rAAV vector or particle is a ssAAV1-PGK-CYP21HA vector that contains a genome with AAV2 ITR sequences and encodes the AAV1 capsid protein. In some embodiments, the rAAV vector or particle is an AAV1-CAG-CYP21, AAV1-PGK-CYP21, AAV1-CBA-CYP21, or AAV1-CB6-CYP21 vector. In other embodiments, the rAAV vector or rAAV particle comprises an AAV5 capsid and a nucleic acid molecule comprising human 21OH cDNA; a CAG, PGK, CBA or CB6 promoter; and, optionally, one or two AAV2 ITR sequences.

[0054] In one embodiment, the rAAV vector or particle is a ssAAV5-PGK-CYP21HA vector that contains a genome with AAV2 ITR sequences and encodes an AAV5 capsid protein. In some embodiments, the rAAV vector or particle is an AAV5-CAG-CYP21, AAV5-PGK-CYP21, AAV5-CBA-CYP21, or AAV5-CB6-CYP21 vector. In yet other embodiments, the rAAV vector or particle is an AAV6 vector that includes a human 21OH cDNA; a CAG, PGK, CBA, or CB6 promoter; and optionally one or two AAV2 ITR sequences. In some embodiments, the rAAV vector or particle is an AAV6-CAG-CYP21, AAV6-PGK-CYP21, AAV6-CBA-CYP21, or AAV6-CB6-CYP21 virus. In further embodiments, the rAAV vector or particle comprises an AAV8 capsid and a nucleic acid molecule comprising human 21OH cDNA; a CAG, PGK, CBA or CB6 promoter; and optionally one or two AAV2 ITR sequences. In some embodiments, the rAAV vector or particle is an AAV8-CAG-CYP21, AAV8-PGK-CYP21, AAV8-CBA-CYP21, or AAV8-CB6-CYP21 virus. In some embodiments, the rAAV vector or particle is an AAV9 vector comprising human 21OH cDNA; a CAG, PGK, CBA or CB6 promoter; and optionally one or two AAV2 ITR sequences. In some embodiments, the rAAV vector or particle is an AAV9-CAG-CYP21, AAV9-PGK-CYP21, AAV9-CBA-CYP21, or AAV9-CB6-CYP21 vector. In additional embodiments, the rAAV vector or particle comprises an AAV10 capsid and a nucleic acid molecule comprising a human 21OH cDNA; a CAG, PGK, CBA, or CB6 promoter; and, optionally, one or two AAV2 ITR sequences.In some embodiments, the rAAV vector or particle is an AAV10-CAG-CYP21, AAV10-PGK-CYP21, AAV10-CBA-CYP21, or AAV10-CB6-CYP21 virus. In some embodiments, the rAAV vector or particle comprises an AAV10 capsid and a nucleic acid molecule comprising a human 21OH cDNA; a CAG, PGK, CBA, or CB6 promoter; and, optionally, one or two AAV2 ITR sequences.

[0055] In one embodiment, the rAAV vector or particle is an AAVrh10-CAG-CYP21A2-HA virus that contains a genome with AAV2 ITR sequences and encodes a rh10 capsid protein. In some embodiments, the rAAV vector or particle is an AAVrh10-CAG-CYP21, AAVrh10-PGK-CYP21, AAVrh10-CBA-CYP21, or AAVrh10-CB6-CYP21 virus. In any of these embodiments, the promoter may comprise or consist of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49, or a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In any of these embodiments, the rAAV vector may include a Kozak sequence. In some embodiments, the Kozak sequence may include or be transcribed to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47. In any of these embodiments, the rAAV vector may further include a HBVPRE or WPRE sequence (e.g., SEQ ID NO:51).

[0056] In some embodiments, the rAAV vector or particle is an AAV5 serotype vector or particle that contains a codon-optimized nucleotide sequence encoding human 21OH under the control of a CBA promoter and with or without a further miR-122 binding sequence, and includes a Kozak sequence. In one embodiment, the rAAV vector or particle is an AAV5-CBA-Kozak-COhCYP21-miR122 vector that includes a CBA promoter, a Kozak sequence, a miR-122 miRNA binding site for detargeting (Thakral and Ghoshal, Curr. Gene Ther. 2015;15(2):142-150), and a codon-optimized (CO) human CYP21 transgene (SEQ ID NO:50). In some embodiments, the rAAV vector or particle is an AAV5-CBA-Kozak-hCYP21 vector, an AAV5-CBA-Kozak-hCYP21-miR122 vector, or an AAV5-CBA-Kozak-COhCYP21 vector. In any of these embodiments, the codon-optimized nucleotide sequence may comprise SEQ ID NO:50, and the Kozak sequence may comprise or be transcribed to SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47. In any of these embodiments, the rAAV vector may further comprise a HBVPRE sequence or a WPRE sequence (e.g., SEQ ID NO:51).

[0057] In some embodiments, an rAAV vector or particle comprises a capsid ("Cap") protein (e.g., including vp1, vp2, vp3 and hypervariable regions), a viral replication ("Rep") protein (e.g., rep78, rep68, rep52, or rep40), and / or a sequence encoding one or more such proteins. These AAV components may be readily utilized in a variety of vector systems and host cells. Such components may be used alone, in combination with other AAV serotype sequences or components, or in combination with elements derived from non-AAV viral sequences. As used herein, an artificial AAV serotype includes, but is not limited to, an AAV having a capsid protein that does not occur in nature. Such artificial capsids may be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence that may be derived from a different selected AAV serotype, from non-contiguous portions of the same AAV serotype, from a non-AAV viral source, or from a non-viral source. The artificial AAV serotype may be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotype vectors in which the capsid of one AAV is replaced with a heterologous capsid protein are useful in the present disclosure. In one embodiment, the AAV is AAV2 / 5. In another embodiment, the AAV is AAV2 / 8. See, e.g., Mussolino et al., Gene Therapy, 18(7):637-645 (2011); Rabinowitz et al., J. Virol, 76(2):791-801 (2002).

[0058] In some embodiments, vectors useful in the compositions and methods described herein contain at least a sequence encoding a selected AAV serotype capsid or a fragment thereof. In some embodiments, useful vectors contain at least a sequence encoding a selected AAV serotype rep protein or a fragment thereof. Optionally, such vectors may contain both an AAV cap protein and a rep protein. In vectors in which both an AAV rep and cap are provided, the AAV rep sequence and the AAV cap sequence can both be of one serotype. Alternatively, a vector may be used in which the rep sequence is from one AAV serotype and the cap sequence is from a different AAV serotype. In one embodiment, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell and a vector). In another embodiment, the rep sequences are fused in frame to a cap sequence of a different AAV serotype to form a chimeric AAV vector, such as AAV2 / 8, as described in U.S. Pat. No. 7,282,199, which is incorporated by reference herein in its entirety.

[0059] A suitable rAAV can be produced by culturing a host cell that contains a nucleic acid sequence encoding an AAV serotype capsid protein or fragment thereof as defined herein; a functional rep gene; a nucleic acid molecule composed of at least an AAV inverted terminal repeat (ITR) and 21OH (CYP21A2) nucleic acid sequence; and sufficient helper functions to allow packaging of the nucleic acid molecule into an AAV capsid protein. In some aspects, the disclosure provides a host cell that includes an rAAV vector or rAAV particle disclosed herein. The components required to be present in the host cell to package the rAAV vector into an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the required components using a variety of methods. Most preferably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein above in the discussion of suitable regulatory elements for use with non-AAV nucleotide sequences, i.e., 21OH. In yet another alternative, the selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, stable host cells may be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter. Still other stable host cells may be generated.

[0060] The rAAV vector, rep sequences, cap sequences, and helper functions required to generate the rAAV of the present disclosure may be delivered to the packaging host cell in the form of any genetic element that transmits the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of the present disclosure, including the generation of rAAV particles, are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY; Fisher et al., J. Virol., 70:520-532 (1993); and U.S. Patent No. 5,478,745, each of which is incorporated herein by reference in its entirety.

[0061] In one aspect, the disclosure provides a method for producing rAAV particles, the method comprising culturing a host cell containing: (a) a nucleic acid molecule comprising or consisting of a rAAV vector genome expressing a 21OH as described herein; (b) a nucleic acid molecule encoding AAV rep; (c) a nucleic acid molecule encoding at least one AAV capsid protein; and (d) sufficient helper functions for packaging the rAAV vector genome into an rAAV particle.

[0062] The rAAV particles of the present disclosure may be purified by various methods. In one embodiment, the rAAV virus may be purified by anion exchange chromatography. See, for example, US Patent Publication No. 2018 / 0163183 A1. Further details regarding the construction and characterization of the AAV vectors and AAV particles disclosed herein are described in International Patent Publication No. WO2019 / 143803, which is incorporated herein by reference in its entirety for all purposes.

[0063] In some embodiments, a composition comprising a non-replicating recombinant AAV serotype 5 (AAV5) vector containing an expression cassette for a human CYP21A2 transgene disclosed herein has the design of the AAV-hCYP21-Opt vector shown in Figure 4. In some embodiments, the transgene sequence packaged into rAAV5 particles is a single stranded DNA composed of the following elements listed from 5' to 3': AAV 5' inverted terminal repeat (ITR), AAV packaging signal (Ψ), cytomegalovirus enhancer, chicken β-actin promoter containing a canonical Kozak sequence with splice donor and rabbit β-globin intron with splice acceptor, codon-optimized human 21-hydroxylase (21-OH) cDNA of SEQ ID NO:50, rabbit β-globin polyA signal, and AAV 3' inverted terminal repeat ITR.

[0064] In some embodiments, the expression cassette of the rAAV vector comprises or consists of the nucleic acid sequence of SEQ ID NO:52. In some embodiments, the expression cassette of the rAAV vector comprises a 5' ITR comprising the nucleic acid sequence of SEQ ID NO:53. In some embodiments, the expression cassette of the rAAV vector comprises a CMVIE enhancer comprising the nucleic acid sequence of SEQ ID NO:54. In some embodiments, the expression cassette of the rAAV vector comprises a CBA promoter comprising the nucleic acid sequence of SEQ ID NO:55. In some embodiments, the expression cassette of the rAAV vector comprises an intron comprising the nucleic acid sequence of SEQ ID NO:56. In some embodiments, the expression cassette of the rAAV vector comprises a codon-optimized hCYP21 comprising the nucleic acid sequence of SEQ ID NO:57. In some embodiments, the expression cassette of the rAAV vector comprises a β-globin poly A comprising the nucleic acid sequence of SEQ ID NO:58. In some embodiments, the expression cassette of the rAAV vector comprises a 3' ITR comprising the nucleic acid sequence of SEQ ID NO:59.

[0065] Pharmaceutical Compositions The rAAV vector or rAAV particle of the present disclosure can be incorporated into a pharmaceutical composition suitable for administration. In one aspect, the present disclosure provides a pharmaceutical composition comprising a rAAV vector or rAAV particle disclosed herein (e.g., a rAAV particle comprising a nucleic acid sequence encoding 21OH) and a pharma- ceutically acceptable carrier, diluent or excipient. As used herein, the term "pharma- ceutically acceptable" refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using established routes. Molecular entities and compositions approved by the U.S. Federal or U.S. State regulatory agencies for use in animals, and more specifically in humans, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias are considered "pharma- ceutically acceptable". As used herein, the term "pharma-ceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in this field, and is incorporated herein by reference.Some examples of such carriers or diluents include, but are not limited to, water, saline, buffered saline, Ringer's solution, glucose solution, 5% human serum albumin, and other buffers for maintaining pH at an appropriate physiological level, such as HEPES.Except in the case where any medium or agent is incompatible with the active compound, its use in the composition is contemplated.Supplementary active compounds can also be incorporated into the composition.

[0066] Throughout this description, "vg" may refer to the "viral genome" or the "vector genome."

[0067] Examples of pharmaceutical compositions and delivery systems that may be used to administer the rAAV disclosed herein can be found in Remington: The Science and Practice of Pharmacy, (2003), 20 thed.,Mack Publishing Co.,Easton,Pa.;Remington's Pharmaceutical Sciences.(1990),18 th ed.,Mack Publishing Co.,Easton,Pa.;The Merck Index.(1996),12 th ed.,Merck Publishing Group,Whitehouse,NJ;Pharmaceutical Principles of Solid Dosage Forms.(1993),Technomic Publishing Co.,Inc.,Lancaster,Pa.;Ansel and Stoklosa,Pharmaceutical Calculations.(2001),11 th ed.,Lippincott Williams & Wilkins,Baltimore,Md.;and Poznansky et al.,Drug Delivery Systems.(1980),Refer to RLJuliano,ed.,Oxford,NY,pp.253-315.

[0068] The pharmaceutical composition of the present disclosure may be formulated to suit its intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous administration or infusion. Infusion may include direct injection into the adrenal gland via laparotomy or laparoscopy, or into the adrenal artery or vein via catheterization. Solutions or suspensions used for parenteral (e.g., intravenous or via injection) application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0069] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0070] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components required from above-listed.For the preparation of sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient plus any additional components from its solution that has been previously sterilized and filtered.

[0071] For injection, the pharma- ceutically acceptable carrier can be a liquid. Exemplary physiologically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate-buffered saline. A variety of such known carriers are provided in U.S. Patent No. 7,629,322, the entirety of which is incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. In one embodiment, the carrier includes TWEEN® (polysorbate). For long-term storage of rAAV, the rAAV may be frozen, for example, in the presence of glycerol or TWEEN® (polysorbate) 20.

[0072] It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined amount of active agent (e.g., rAAV) calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are determined by and directly depend on the unique characteristics of the active agent (e.g., rAAV) and the particular therapeutic effect to be achieved, as well as the inherent limitations of the art of compounding such active agents (e.g., rAAV) for the treatment of individuals. The unit dosage forms may include liquid compositions, or compositions in lyophilized or lyophilized form, for example, in ampoules and vials; for example, a sterile liquid carrier can be added prior to administration or delivery in vivo. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector (e.g., rAAV) sequences, plasmids, vector genomes, recombinant viral particles (e.g., rAAV), and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0073] Pharmaceutical compositions containing a rAAV vector or rAAV particle comprising a nucleic acid sequence encoding 21OH can be included in a container, pack, or dispenser together with instructions for administration.

[0074] In some embodiments, the composition is 5.0×10 13 vg / mL (3.0 × 10 13 ~7.0×10 13 The composition is a single-dose, preservative-free, sterile, opaque, colorless IV injection solution of a non-replicating recombinant AAV5 vector disclosed herein at a target concentration of 1000 mg / mL (1000 mg / mL). In some embodiments, the composition contains 10 mM sodium phosphate, 180 mM sodium chloride, 0.005% w / v poloxamer 188, and water for injection. In some embodiments, the composition is filled into 5 mL Crystal Zenith® (CZ, cyclic olefin polymer) vials with a nominal fill volume of 2.5 mL and stored at or below -60°C.

[0075] In one aspect, the disclosure provides a kit comprising a rAAV vector, or a particle comprising the same, wherein the rAAV vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, the non-AAV nucleotide sequence being operably linked to a promoter; and a therapeutically effective amount is about 10 12 vg / kg~about 10 14 In some embodiments, the therapeutically effective amount is in the range of about 1.5×10 13 In some embodiments, the therapeutically effective amount is about 3×10 13 In some embodiments, the therapeutically effective amount is about 6×10 13 vg / kg. In some embodiments, the kit further comprises instructions for administering the rAAV vector to a subject. In some embodiments, the kit further comprises instructions for administering the rAAV vector intravenously by direct injection into the adrenal gland via laparotomy or laparoscopy, or by injection into the adrenal artery or vein via catheter insertion into the subject.

[0076] In another aspect, the disclosure provides a unit dose comprising a rAAV vector, or a particle comprising the same, wherein the rAAV vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, the non-AAV nucleotide sequence being operably linked to a promoter; a therapeutically effective amount is 10 12 vg / kg~about 10 14 In some embodiments, the therapeutically effective amount is in the range of about 1.5×10 13 In some embodiments, the therapeutically effective amount is about 3×10 13 In some embodiments, the therapeutically effective amount is about 6×10 13 vg / kg. In some embodiments, the unit dose comprises a liquid formulation. In some embodiments, the unit dose is configured to be administered intravenously to a subject by direct injection into the adrenal gland via laparotomy or laparoscopy, or by injection into the adrenal artery or vein via catheterization.

[0077] In some embodiments of the above aspects, the 21OH protein is a human 21OH protein. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the 21OH protein comprises or consists of a cDNA for human 21OH (CYP21A2). In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the 21OH protein comprises or consists of a codon-optimized nucleotide sequence. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the 21OH protein comprises or consists of SEQ ID NO:50. In some embodiments of the above aspects, the non-AAV nucleotide sequence encoding the 21OH protein encodes an amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. In some embodiments of the above aspects, the promoter directs expression of the 21OH protein in the host cell. In some embodiments of the above aspects, the host cell is an adrenal cell or an adrenal cortex cell. In some embodiments of the above aspects, the promoter is a cytomegalovirus / β-actin hybrid promoter, a PGK promoter, or a promoter specific for expression in adrenal cortex cells. In some embodiments of the above aspects, the cytomegalovirus / β-actin hybrid promoter is a CAG, CB6, or CBA promoter. In some embodiments of the above aspects, the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49. In some embodiments of the above aspects, the ITR is an ITR of the serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74. In some embodiments of the above aspects, the rAAV is of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype.In some embodiments of the above aspects, the rAAV is an AAV5 serotype. In some embodiments of the above aspects, the nucleic acid molecule further comprises a Kozak sequence. In some embodiments of the above aspects, the nucleic acid molecule further comprises a miR-122 binding site.

[0078] Methods of Use of Recombinant AAV Vectors and Particles The present disclosure provides methods that include the use of AAV (e.g., recombinant AAV) vectors, such as in the treatment of a subject in need thereof. The methods provided herein include, for example, administering to a subject a therapeutically effective amount of any one of the rAAV or compositions disclosed herein.

[0079] In certain embodiments, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a rabbit, a guinea pig, a hamster, a mouse, or a rat. In certain embodiments, the subject is a human. The human subject may be a human female or a human male. In some embodiments, the subject is a human who has previously undergone a hormone therapy program. In some embodiments, the subject is a human infant. In certain cases, the subject is a human infant about 1 month old, about 2 months old, about 3 months old, about 4 months old, about 5 months old, about 6 months old, about 7 months old, about 8 months old, about 9 months old, about 10 months old, about 11 months old, or about 1 year old. In some embodiments, the subject is a human infant less than 3 months old, less than 6 months old, less than 9 months old, less than 1 year old, or less than 18 months old.

[0080] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk for or suffering from a disease, disorder, or condition that can be treated or ameliorated by a rAAV comprising a nucleic acid sequence encoding 21OH or a composition comprising such a rAAV provided herein. A patient or subject in need may be a patient or subject diagnosed with a disease associated with dysfunction of 21OH, such as 21OHD, e.g., CAH. The subject may have a mutation or dysfunction in the 21OH gene or protein. "Subject" and "patient" are used interchangeably herein.

[0081] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent, e.g., rAAV, that is sufficient to reduce or ameliorate the severity and / or duration of a disorder, e.g., 21OHD or one or more symptoms thereof, prevent the progression of a disorder, cause regression of a disorder, prevent the recurrence, onset, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic effect(s) or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent). An effective amount of rAAV may, for example, increase the expression of 21OH and / or alleviate to some extent one or more symptoms associated with 21OHD.

[0082] The present disclosure provides methods and uses of rAAVs comprising nucleic acid molecules encoding 21OH as described herein to provide a therapeutic effect to a subject having a disorder or disease characterized by a deficiency or dysfunction of 21OH. In some embodiments, the methods include administering a therapeutically effective amount of the rAAV or composition described herein to a subject in need of administering the rAAV or composition, thereby treating the disorder or disease characterized by a deficiency or dysfunction of 21OH in the subject.

[0083] In some cases, the present disclosure provides a method of expressing 21OH in a subject in need of expressing 21OH, comprising administering to the subject a therapeutically effective amount of a rAAV particle described herein or a pharmaceutical composition described herein, thereby expressing 21OH in the subject. In certain embodiments, the present disclosure provides a method of increasing expression of 21OH in a subject in need of expressing 21OH, comprising administering to the subject a therapeutically effective amount of a rAAV particle described herein or a pharmaceutical composition comprising the particle, thereby increasing expression of 21OH in the subject. In some embodiments, the methods disclosed herein result in expression of 21OH or increase expression of 21OH in the adrenal gland (e.g., adrenal cortex or adrenal medulla), liver or ovary of the subject. In certain embodiments, the methods disclosed herein result in expression of 21OH or increase expression of 21OH in the adrenal stem cell (e.g., adrenal cortex stem cell) or adrenal progenitor cell of the subject. In certain embodiments, the methods disclosed herein result in expression of 21OH or increase expression of 21OH in the liver or ovary of the subject.

[0084] The present disclosure provides a method of treating a subject with 21-hydroxylase deficiency (21OHD), the method comprising administering to the subject a therapeutically effective amount of a rAAV particle or a pharmaceutical composition comprising the particle as described herein, thereby treating 21OHD in the subject. The present disclosure provides a method of increasing cortisol and / or aldosterone levels in a subject in need of increased cortisol and / or aldosterone levels, the method comprising administering to the subject a therapeutically effective amount of a rAAV particle or a composition as disclosed herein, thereby increasing cortisol and / or aldosterone levels in the subject. The present disclosure provides a method of decreasing progesterone, 17-OHP, renin and / or androstenedione (A4) levels in a subject in need of decreased progesterone, 17-OHP, renin and / or androstenedione (A4) levels, the method comprising administering to the subject a therapeutically effective amount of a rAAV particle or a composition as disclosed herein, thereby decreasing progesterone, 17-OHP, renin and / or androstenedione (A4) levels in the subject.

[0085] The present disclosure further provides a method of treating, alleviating, ameliorating, slowing the progression, or preventing symptoms of 21OHD (or CAH) in a subject having 21OHD (or congenital adrenal hyperplasia, CAH), comprising administering to the subject a therapeutically effective amount of a rAAV particle described herein or a pharmaceutical composition comprising the particle, thereby treating, alleviating, ameliorating, slowing the progression, or preventing symptoms of 21OHD in the subject. Non-limiting examples of symptoms of 21OHD (or CAH) include virilization of genitalia and muscle mass, salt loss and dehydration during infancy, sexual dysfunction (classic type) and hirsutism, acne, and reduced fertility (classic and non-classic types). In male patients, non-limiting examples of symptoms of 21OHD (or CAH) include short stature and early virilization. The Prader classification system is used to measure the degree of virilization of the human genitalia. In some embodiments, the subject (e.g., a female subject) treated by the methods of the present disclosure has an external genitalia abnormality according to the Prader classification (e.g., the subject has Prader type IV or V 21OHD (or CAH).

[0086] In some embodiments, the subject is in need of 21OH expression. In some embodiments, the subject has 21OH deficiency (21OHD). In some embodiments, the subject (e.g., a female subject) has Prader external genitalia anomalies (e.g., the subject has Prader type IV or V 21OHD (or CAH). In some embodiments, the subject has congenital adrenal hyperplasia (CAH). In some embodiments, the CAH is classical CAH. In some embodiments, the subject is in need of cortisol and / or aldosterone. In some embodiments, the subject has cortisol deficiency and / or aldosterone deficiency. In some embodiments, the subject has excess progesterone, 17-OHP, renin, and / or androstenedione (A4). In some embodiments, the subject has excess progesterone, 17-OHP, renin, and / or androstenedione (A4) in the blood and / or urine.

[0087] In some embodiments, the subject has a value of about 5 times to about 10 times the ULN (upper limit of normal) for 17-OHP. In some embodiments, the subject has an androstenedione (A4) value above the ULN. In some embodiments, the subject has an 11-oxo or 11-keto sterol (e.g., 11-keto testosterone) value above the ULN. In some embodiments, the subject is receiving a stable regimen of oral hydrocortisone as the sole glucocorticoid maintenance therapy at a total daily dose of about 30 milligrams per day (mg / day) of hydrocortisone. In some embodiments, the subject is receiving a stable regimen of oral hydrocortisone as the sole glucocorticoid maintenance therapy at a total daily dose of about 10 mg / day to about 50 mg / day of hydrocortisone.

[0088] In some embodiments, the method further comprises selecting a subject having 21OHD before the administration step. In some embodiments, the subject may be screened and identified or diagnosed (e.g., by genetic or physiological testing) as having 21OHD, even if the subject does not have one or more symptoms of the disease. In other embodiments, the subject has one or more symptoms of 21OHD. In certain embodiments, the subject has a mutation in the CYP21A2 gene. In one embodiment, the subject has a loss-of-function mutation in the CYP21A2 gene. In certain embodiments, the subject has a defect in the CYP21A2 gene caused by gene conversion or recombination between the functional CYP21A2 gene and the closely linked non-functional CYP21A1P pseudogene.

[0089] In some embodiments, the level of cortisol and / or aldosterone is higher in the subject after administration of the rAAV or composition as disclosed herein compared to before administration of the rAAV or composition. In some embodiments, the level of cortisol and / or aldosterone is higher in the subject after administration of the rAAV or composition as disclosed herein compared to a control subject with 21-hydroxylase deficiency (21OHD) who is not administered the rAAV or composition as disclosed herein. In some embodiments, the level of progesterone, 17-OHP, renin and / or androstenedione (A4) is lower in the subject after administration of the rAAV or composition as disclosed herein compared to before administration of the rAAV or composition. In some embodiments, the level of progesterone, 17-OHP, renin and / or androstenedione (A4) is lower in the subject after administration of the rAAV or composition as disclosed herein compared to a control subject with 21-hydroxylase deficiency (21OHD) who is not administered the rAAV or composition as disclosed herein.

[0090] In some embodiments of the methods disclosed herein, hepatotoxic substances (e.g., ketamine, halogenated anesthetics) are not administered to the subject in combination with the rAAV or compositions disclosed herein. In some embodiments of the methods disclosed herein, hepatotoxic substances are not administered for at least 12 weeks after administration of the rAAV or compositions disclosed herein. In some embodiments of the methods disclosed herein, acetaminophen (and other similar agents) are not administered to the subject in combination with the rAAV or compositions disclosed herein. In some embodiments of the methods disclosed herein, substances that inhibit or induce tacrolimus metabolism via the CYP3A or CYP3A4 pathways, including grapefruit juice, are not administered to the subject in combination with the rAAV or compositions disclosed herein.

[0091] Mode of administration In any of the therapeutic methods described herein, the rAAV particles comprising a nucleic acid molecule encoding 21OH, or a pharmaceutical composition comprising the particles, may be administered to a subject by any means that introduces the rAAV particles into the adrenal cortex vasculature or into the adrenal cortex itself. In some embodiments, the rAAV particles comprising a nucleic acid molecule encoding 21OH, or a pharmaceutical composition comprising the particles, may be administered to a subject systemically. In some embodiments, the rAAV particles comprising a nucleic acid molecule encoding 21OH, or a pharmaceutical composition comprising the particles, may be administered to a subject intravenously; by direct injection into the adrenal gland via laparotomy or laparoscopy; by injection into the adrenal artery or vein via catheterization. In some embodiments, the direct injection into the adrenal gland is a direct injection into the adrenal cortex.

[0092] In some embodiments, rAAV particles comprising a nucleic acid molecule encoding 21OH as described herein, or pharmaceutical compositions comprising such particles, may be used to treat subjects suffering from congenital adrenal hyperplasia (CAH). Deficiency of 21OH often causes CAH, a family of genetic disorders that affect the adrenal glands. CAH may be present in a subject in severe or mild forms. Severe or classical forms, referred to as classical CAH, are usually detected in the neonatal or early childhood period. Milder forms, referred to as non-classical CAH (NCAH or NCCAH) or late-onset CAH, may cause symptoms any time from infancy to adulthood (see, e.g., Kurtoglu et al., J. Clin. Res. Pediatr. Endocrinol, 9(1):1-7(2017)). The rAAV of the present disclosure may be used to treat subjects with classical or non-classical CAH. Subjects with classic CAH may experience fetal virilization of the external genitalia, reduced or absent production of glucocorticoids and mineralocorticoids, and / or excessive production of androgens.Subjects with nonclassical CAH may experience fetal virilization and increased production of androgens without deficiency of cortisol or aldosterone.

[0093] Cortisol is a steroid produced by the adrenal glands. Cortisol is used by the body in response to physical and emotional stress and to maintain an adequate energy supply and blood sugar levels. The adrenal glands are controlled by the pituitary gland, a small pea-sized gland at the base of the brain. In healthy individuals, when there is insufficient cortisol present in the bloodstream, the pituitary gland releases adrenocorticotropic hormone (ACTH). ACTH stimulates the adrenal glands to produce more cortisol. However, individuals with CAH have insufficient amounts of 21OH, which is needed to convert the precursor 17-hydroxyprogesterone (17-OHP) to cortisol. As a result, the pituitary gland continues to sense the need for cortisol and releases more ACTH. This leads to excess 17-OHP, which is converted by the adrenal glands into excess androgens (masculinizing steroid hormones). Thus, subjects may be diagnosed with CAH by measuring increased circulating levels of the affected steroid hormone. Newborn screening for 21OHD is usually accomplished using 17-OHP measurements. Additionally, subjects with CAH may be monitored by tracking circulating levels of 17-OHP. Thus, in some embodiments, circulating levels of the affected steroid hormone may be measured in a subject with CAH before, during, and / or after treatment with a rAAV particle comprising a nucleic acid molecule encoding 21OH as described herein or a pharmaceutical composition comprising such a particle. Circulating levels of 17-OHP in a subject may be used to diagnose the subject and inform decisions about whether to initiate or continue treatment of the subject with a rAAV particle or pharmaceutical composition as described herein.

[0094] In some embodiments, the methods disclosed herein have one or more of the following effects: (1) reducing or eliminating the dependency on exogenous glucocorticoids (GCs) by restoring physiological endogenous cortisol biosynthesis, thereby reducing the sequelae of supraphysiological GC therapy; (2) reducing the hyperandrogenism associated with 21-OHD without increasing exogenous GC dosage, thereby reducing the sequelae of hyperandrogenism; (3) reducing the risk of associated life-threatening adrenal crisis by restoring endogenous cortisol and / or aldosterone biosynthesis; and (4) reducing the patient burden and non-compliance associated with daily administration of supraphysiological GC and / or mineralocorticoid (MC) replacement therapy. Thus, in some embodiments, the methods disclosed herein help alleviate adrenal hormone deficiency in CAH, thereby ameliorating the underlying pathobiology of the disease.

[0095] The present disclosure further contemplates the use of the pharmaceutical agent described herein (e.g., rAAV or a pharmaceutical composition comprising rAAV) in the manufacture of a medicament for treating a disorder or disease characterized by 21OH dysfunction or deficiency in a subject. The present disclosure also includes the use of the pharmaceutical agent described herein (e.g., rAAV or a pharmaceutical composition comprising rAAV) for treating a disorder or disease characterized by 21OH dysfunction or deficiency in a subject. Dosage

[0096] The composition may be delivered in a volume of about 50 microliters (μL) to about 1 milliliter (mL), including all values ​​within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume is about 50 μL. In another embodiment, the volume is about 70 μL. In another embodiment, the volume is about 100 μL. In another embodiment, the volume is about 125 μL. In another embodiment, the volume is about 150 μL. In another embodiment, the volume is about 175 μL. In yet another embodiment, the volume is about 200 μL. In another embodiment, the volume is about 250 μL. In another embodiment, the volume is about 300 μL. In another embodiment, the volume is about 450 μL. In another embodiment, the volume is about 500 μL. In another embodiment, the volume is about 600 μL. In another embodiment, the volume is about 750 μL. In another embodiment, the volume is about 850 μL. In another embodiment, the volume is about 1000 μL.

[0097] In some embodiments, the effective concentration of a rAAV carrying a nucleic acid sequence encoding a particular transgene (e.g., 21OH) under the control of a promoter sequence is about 10 per milliliter. 8 ~about 10 13 Vector genome (vg / mL). For example, rAAV infectious units may be measured as described in McLaughlin et al., J. Virol., 62:1963 (1988). In some embodiments, the concentration is about 1.5×10 9 vg / mL ~ approx. 1.5×10 12 In some embodiments, the concentration is about 1.5×10 9 vg / mL ~ approx. 1.5×10 11 In one embodiment, the effective concentration is about 1.5×10 10 In another embodiment, the effective concentration is about 1.5×10 11 In another embodiment, the effective concentration is about 2.8×10 11 In yet another embodiment, the effective concentration is about 1.5×10 12In a further embodiment, the effective concentration is about 1.5×10 13 In some embodiments, the concentration is about 10 vg / mL, including all values ​​and subranges therebetween. 13 vg / mL ~ approx. 7.0×10 13 vg / mL, e.g., about 2×10 13 vg / mL, approximately 3×10 13 vg / mL, approximately 4×10 13 vg / mL, approximately 5×10 13 vg / mL, approximately 6×10 13 vg / mL, approximately 7×10 13 In some embodiments, the concentration is about 5×10 13 vg / mL.

[0098] In some embodiments, the lowest effective concentration of the virus is utilized to reduce the risk of undesirable effects such as toxicity or adverse immune response.Other dosages within these ranges may be selected by the attending physician, taking into account the physical condition of the subject (e.g., human) being treated, the subject's age, the specific 21OH deficiency, and the degree of the disorder occurring if progressive.

[0099] In some embodiments, the rAAV vector or particle comprising a nucleic acid sequence encoding 21OH is administered at a dose of about 10 per kg of subject body weight. 11 ~about 10 14 vg / kg, e.g., about 10 per kg of subject body weight 12 vg / kg~about 10 14 In some embodiments, the rAAV vector or particle comprising a nucleic acid sequence encoding 21OH is administered to the subject at a dose ranging from about 1.5×10 13 vg / kg, 3 × 10 13 vg / kg, or 6 × 10 13 The subject is administered a dose of 1000 mg / kg.

[0100] In some embodiments, a therapeutically effective amount is about 10 to 20 mg, including all values ​​and subranges therebetween. 12 vg / kg~about 1014 vg / kg range, e.g., about 5×10 12 vg / kg, approx. 10 13 vg / kg, or approximately 5 × 10 13 In some embodiments, the therapeutically effective amount is about 10 vg / kg, including all values ​​and subranges therebetween. 13 vg / kg~about 10 14 vg / kg range, e.g., about 1.5×10 13 vg / kg, 2×10 13 vg / kg, 2.5×10 13 vg / kg, 3 × 10 13 vg / kg, 3.5×10 13 vg / kg, 4 × 10 13 vg / kg, 4.5×10 13 vg / kg, 5×10 13 vg / kg, 5.5×10 13 vg / kg, 6×10 13 vg / kg, 6.5×10 13 vg / kg, 7×10 13 vg / kg, 7.5×10 13 vg / kg, 8×10 13 vg / kg, 8.5×10 13 vg / kg, 9×10 13 vg / kg, or 9.5 × 10 13 In some embodiments, the therapeutically effective amount is about 1.5×10 13 In some embodiments, the therapeutically effective amount is about 3×10 13 In some embodiments, the therapeutically effective amount is about 6×10 13 vg / kg.

[0101] In some embodiments, the subject is administered a single dose of a rAAV vector, rAAV particle, or composition disclosed herein. In some embodiments, the subject is administered multiple doses, e.g., two, three, four, or five doses, of a rAAV vector, rAAV particle, or composition disclosed herein.

[0102] The compositions disclosed herein may be administered to a subject at any frequency. In some embodiments, the compositions may be administered to a subject once a day or more than once a day. In some embodiments, the compositions may be administered to a subject 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a day. In some embodiments, the compositions may be administered to a subject daily, every other day, every 3, 4, 5, or every 6 days. In some embodiments, the compositions may be administered to a subject weekly, every other week, or every 3 weeks. In some embodiments, the compositions may be administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months. In some embodiments, the compositions may be administered to a subject annually. In some embodiments, the compositions may be administered to a subject every 1, 2, 3, 4, 5, 10, 15, or 20 years. In certain embodiments, the compositions may be administered to a subject only once during the subject's lifetime.

[0103] Combination with immunosuppressants In some embodiments, any one of the methods disclosed herein further comprises administering a therapeutically effective amount of an immunosuppressant. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a rAAV or composition disclosed herein and a therapeutically effective amount of an immunosuppressant. The present disclosure provides a method of treatment comprising administering to a subject a therapeutically effective amount of a rAAV or composition disclosed herein and a therapeutically effective amount of an immunosuppressant.

[0104] In some embodiments, the immunosuppressant is administered prior to, concurrently with, and / or after administration of the rAAV vector. In some embodiments, the immunosuppressant is administered prior to administration of the rAAV vector. In some embodiments, the immunosuppressant is administered at least 12 hours prior to administration of the rAAV vector. In some embodiments, the immunosuppressant is administered about 2 days prior to administration of the rAAV vector.

[0105] In some embodiments, the immunosuppressant is a non-glucocorticoid immunosuppressant. In some embodiments, the immunosuppressant is an inhibitor of calcineurin. In some embodiments, the immunosuppressant is cyclosporine, tacrolimus, sirolimus, everolimus, zotarolimus, or any combination thereof. In some embodiments, the immunosuppressant is tacrolimus. Other non-limiting examples of immunosuppressants include alkylating agents such as nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, folic acid analogs such as methotrexate, purine analogs such as azathioprine and mercaptopurine, pyrimidine analogs such as fluorouracil, protein synthesis inhibitors, cytotoxic antibodies such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, polyclonal antibodies that inhibit T lymphocytes, monoclonal antibodies directed against the IL-2 receptor such as basiliximab or daclizumab, anti-CD3 monoclonal antibodies such as muromonab, opioids, TNF-alpha binding proteins such as infliximab, etanercept, or adalimumab, mycophenolate esters, fingolimod, and myriocin.

[0106] In some embodiments, the immunosuppressant is administered orally. In some embodiments, the therapeutically effective amount of the immunosuppressant is in the range of about 0.005 milligrams per kilogram (mg / kg) to about 0.1 mg / kg, e.g., about 0.01 mg / kg, about 0.015, about 0.02, about 0.025, about 0.03, about 0.035, about 0.04, about 0.045, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, or about 0.1 mg / kg, including all values ​​and subranges therebetween. In some embodiments, the therapeutically effective amount of the immunosuppressant is in the range of about 0.01 mg / kg to about 0.05 mg / kg. In some embodiments, the therapeutically effective amount of the immunosuppressant is about 0.025 mg / kg. In some embodiments, the therapeutically effective amount of the immunosuppressant is administered twice daily.

[0107] In some embodiments, the method includes administering a therapeutically effective amount of a steroid to the subject. In some embodiments, the steroid is a mineralocorticoid. In some embodiments, the steroid is a glucocorticoid. In some embodiments, the steroid is hydrocortisone. In some embodiments, the steroid is administered prior to, concurrently with, and / or after administration of the rAAV vector. In some embodiments, a therapeutically effective amount of the steroid administered to the subject prior to administration of the rAAV vector is greater than a therapeutically effective amount of the steroid administered to the subject after administration of the rAAV vector. EXAMPLES

[0108] Example 1: Rescue of Congenital Adrenal Hyperplasia (CAH) Symptoms in Mice Construction of AAV vectors encoding 21-hydroxylase (21OH) proteins was performed as described in International Patent Publication No. WO2019 / 143803, which is incorporated by reference in its entirety for all purposes.

[0109] H-2 aw18 (Cyp21 - / - ) mice are the only animal model of human CAH due to 21-OHD that mimics key pathophysiological features of the clinical condition of human CAH, i.e., growth retardation leading to postnatal morbidity due to glucocorticoid (GC) and mineralocorticoid (MC) deficiency. An AAV serotype 5 (AAV5) vector (also referred to herein as "BBP-631") containing a codon-optimized human CYP21A2 transgene of SEQ ID NO:50 under the control of the CBA promoter was transfected into 15-week-old mice. - / - Mice were treated with untreated CYP21 and the disease phenotype was maintained for at least 10 weeks. - / -The expression of human CYP21A2 was compared to that of mice. Vector genome copies (VGC) of the human CYP21A2 transgene and mRNA normalized to mouse GAPDH were measured in the adrenal gland and other organs. Adrenal 21-OH protein expression was assessed using nanoLCMSMS proteomics custom-built to detect human 21-OH specific peptides normalized to GAPDH and endogenous mouse 21-OH peptides.

[0110] The results showed that administration of AAV5 containing a functional copy of the human CYP21A2 gene significantly increased the expression of CYP21 - / - We have shown that the treatment resulted in early and sustained disease rescue in mice, accompanied by: (i) a reduction in urinary progesterone levels throughout the 10 weeks, consistent with restoration of 21-hydroxylation of progesterone; (ii) a reduction in renin expression in the kidney, suggesting improved MC function; and (iii) dose-dependent detection of vector genome, human CYP21A2 mRNA, and human 21-OH protein in the adrenal gland. Treated mice showed a dose-dependent weight gain that correlated with adrenal VGC, transgene mRNA levels, and expression of human 21-OH protein. The dose-dependent weight gain and dose-dependent detection of vector genome in the adrenal gland of mice are shown in Figure 1A and Figure 1B, respectively.

[0111] In conclusion, gene therapy of CYP21− / − mice with AAV serotype 5 (AAV5) vectors containing a codon-optimized human CYP21A2 transgene under the control of the CBA promoter was robust and dose-dependent at the molecular level and in correcting the disease phenotype.

[0112] In WT mice, GLP-compliant toxicology studies assessed safety (clinical observations, pathology, hematology, clinical chemistry, histopathology, and immune responses) and biodistribution at 4, 12, and 24 weeks. Vector genome and transgene expression was detected in the adrenal gland and other tissues. In WT C57BL / 6 mice, no test article-related toxicity was observed at any study time point following a single IV dose of BBP-631 at any dose examined. The study was conducted using a 3.0 × 1014 It was well tolerated with a no observed adverse effect level (NOAEL) of 100 vg / kg and demonstrated sustained transgene delivery and expression in the adrenal gland.

[0113] Example 2: Expression of 21-OH in non-human primates (NHP) Sustained dose-dependent expression of human 21-OH protein in the adrenal glands of NHPs administered one dose of an AAV vector (BBP-631) encoding the 21-hydroxylase (21OH) protein is shown in Figure 1B and Figure 1C. The amount of human 21-OH produced, expressed as a percentage of endogenous 21-OH in NHPs, suggests a possible clinically meaningful disease impact in classical CAH patients (Figure 1C). Identification of biologically active doses in mice and NHPs supports the starting dose in a Phase 1 / 2 clinical trial with the disclosed AAV serotype 5 (AAV5) vector containing a codon-optimized human CYP21A2 transgene under the control of the CB6 promoter.

[0114] Biodistribution and safety studies were performed in 2-2.5 year old NHPs (cynomolgus monkeys). After IV administration of AAV5 containing a functional copy of the human CYP21A2 gene, no adverse safety signals were found across all doses for clinical observations, body or organ weights, urinalysis, hematology, serum chemistry, gross pathology, histopathology (including thoracic spinal cord), immunohistochemistry (no CD4 lymphocyte infiltration), or anti-drug reactions (cell-mediated immune responses to AAV5 capsid or human 21-OH). Dose-dependent viral genome copies (VGCs) and stable mRNA transcript expression were detected in the adrenal gland up to 24 weeks. Proteomic methods demonstrated dose-dependent and sustained expression of human 21-OH protein up to 9-24% of endogenous 21-OH levels. Overall, the results indicate that delivery of AAV5 containing a functional copy of the human CYP21A2 gene was well tolerated and sustained expression in the adrenal gland of NHPs for at least 24 weeks.

[0115] Example 3: Phase 1 / 2 Clinical Trial overview The Phase 1 / 2 first-in-human, open-label, dose-escalation study was designed to evaluate the safety, tolerability, and efficacy of an AAV serotype 5 (AAV5) vector containing a codon-optimized human CYP21A2 transgene under the control of the CB6 promoter (also referred to herein as "BBP-631") administered to up to 25 adult participants diagnosed with classical congenital adrenal hyperplasia (CAH) (simple virilization or salt-losing, group 1) or classical salt-losing CAH due to 21-hydroxylase deficiency (21OHD) (group 2) and monitored for 52 weeks after treatment. All participants receiving BBP-631 treatment will be followed for safety and efficacy for an additional four years. In total, all participants will be followed for at least five years from the date of treatment with BBP-631. Figure 2 shows the timeline of the study.

[0116] BBP-631 will be administered undiluted as one single IV infusion via a volumetric infusion pump. The active agent of BBP-631 is a non-replicating recombinant AAV serotype 5 (AAV5) vector containing an expression cassette for the human CYP21A2 transgene. The design of the AAV-hCYP21-Opt vector is shown in Figure 4. The transgene sequence packaged into the rAAV5 particle is a single stranded DNA composed of the following elements listed from 5' to 3': AAV5' inverted terminal repeat (ITR), AAV packaging signal (Ψ), cytomegalovirus enhancer, chicken β-actin promoter containing a canonical Kozak sequence with splice donor and rabbit β-globin intron with splice acceptor, codon-optimized human 21-hydroxylase (21-OH) cDNA, rabbit β-globin polyA signal, and AAV3' inverted terminal repeat ITR. This optimized promoter design, called CAG, aims to maximize transgene expression in host tissues.

[0117] BBP-631 formulation (DP) was 5.0 × 10 13 vg / mL (3.0 × 10 13 ~7.0×10 13BBP-631DP is a preservative-free, sterile, opaque, colorless, single-dose IV injection solution of non-replicating recombinant AAV5 vector at a target concentration of 10 ...

[0118] Screening Period For all participants, the screening period will begin at the time of the first screening assessment. The screening period will be up to 42 days prior to treatment with BBP-631 (Day 0). Eligibility will be determined by evaluation of safety and screening laboratory samples and threshold levels of 17-OHP, androstenedione (A4), adrenocorticotropic hormone (ACTH), and cortisol. To measure variability in 17-OHP, at least three assessments of 17-OHP will be performed, separated by at least 5 days. At the time of 17-OHP assessment, samples will also be taken to assess liver function. During the screening period, participants will continue to take their normally scheduled oral hydrocortisone and, if applicable, mineralocorticoid treatment for CAH. Unless otherwise specified, screening samples will be taken the morning before participants take their normally scheduled first oral hydrocortisone treatment and, if applicable, mineralocorticoid treatment. In addition to 17-OHP and androstenedione (A4) levels, other hormone levels will be evaluated, including cortisol, hormones related to the hypothalamic-pituitary-adrenal (HPA) axis, the hypothalamic-pituitary-gonadal (HPG) axis, other physiological or regulatory functions, renin and aldosterone, and a panel of steroid hormones. Samples will be analyzed by immunoassay, liquid chromatography-tandem mass spectrometry (LC-MS / MS), or both methods, as appropriate. All hormone level measurements will be performed in a central laboratory.

[0119] Baseline, treatment, and follow-up periods The total duration of the baseline, treatment, and follow-up periods is expected to be approximately 52 weeks.

[0120] Baseline (before treatment with BBP-631): Inpatient (days -4 to 0) Baseline levels of other endogenous hormones, quality of life (QOL), skeletal and body composition assessments, as well as biomarkers of bone turnover, hirsutism, ovulation, testicular function, and testicular adrenal rest tumor (TART) volume will be assessed. Unless otherwise specified, assessments will be performed as close to day 0 (dosing) as possible to establish baseline measurements. Samples will be analyzed by immunoassay, LC-MS / MS, or both methods, as appropriate. All hormone level measurements will be performed in a central laboratory.

[0121] On day -3, participants will have serial blood draws to assess cortisol clearance, ACTH, 17-OHP, and androstenedione (A4) levels. Oral hydrocortisone and, where applicable, mineralocorticoid treatment will be withheld after the regularly scheduled morning dose on day -3.

[0122] -On the morning of day 2, prior to their normally scheduled dose of hydrocortisone and mineralocorticoids, if applicable, participants will be sampled for hormone profile, including but not limited to, HPA axis and renin-aldosterone axis. These samples will serve as a baseline assessment of most hormones, including but not limited to, 17-OHP, androstenedione (A4), and endogenous ACTH. Participants will then complete an ACTH stimulation test for morning endogenous cortisol and 17-OHP levels, and will only take their normally scheduled morning dose of hydrocortisone and mineralocorticoids, if applicable. -On the evening of day 2, samples will be taken every 2 hours for 24 hours to establish the participant's diurnal hormone profile by measuring levels of HPA axis hormones, including but not limited to, cortisol, 17-OHP, androstenedione (A4), and ACTH.

[0123] On the morning of Day 2, participants will begin a prophylactic immunosuppression regimen with tacrolimus to prevent or suppress a potential immune response following BBP-631 administration. This tacrolimus regimen will have a starting dose of 0.025 mg / kg taken orally twice daily with a target trough concentration of 5-8 ng / mL. Trough concentrations will be measured simultaneously with selected blood chemistry analytes.

[0124] In the evening of Day -1, after the 24-hour diurnal hormone profile sampling period is completed, participants will resume their normally scheduled hydrocortisone and, if applicable, mineralocorticoid medications.

[0125] Baseline: Treatment with BBP-631 (Day 0) On day 0, prior to administration of BBP-631 and tacrolimus, tacrolimus trough concentrations will be measured and participants will also receive prophylactic antihistamines such as diphenhydramine (IV), hydroxyzine (IM), and chlorpheniramine (IV) to prevent infusion reactions. BBP-631 will be administered as an intravenous (IV) infusion, starting 2-6 hours after the participant's regularly scheduled morning hydrocortisone dose. Participants will receive only a single dose of BBP-631.

[0126] Observation Continue to assess safety, including laboratory, immunogenicity, and vector shedding. Evaluate safety and efficacy data for decisions regarding dose escalation and expansion. Measure tacrolimus trough concentrations according to maintenance prophylactic immunosuppression schedules and as part of a reactive immunosuppression plan, including liver function assessments, if medically indicated.

[0127] The efficacy and durability of treatment with BBP-631 will be evaluated through samples collected in both inpatient and outpatient (home) settings. Samples will be collected for evaluation of endogenous hormones related to the HPA axis, HPG axis, other physiological or regulatory functions, renin and aldosterone, and a panel of steroid hormones. Unless otherwise specified, samples will be collected in the morning before participants take their normally scheduled morning dose of hydrocortisone and mineralocorticoids, if applicable. Samples will also be collected before and after morning exogenous ACTH stimulation. Participants will undergo serial blood draws to assess cortisol clearance, ACTH, 17-OHP, and androstenedione (A4) levels. Additional markers may also be evaluated, including 11-oxo / 11-keto sterols such as 11-ketotestosterone. Samples will be analyzed by immunoassay, LC-MS / MS, or both methods, as appropriate.

[0128] Quality of life assessments will be measured via the Short-Form (36) Health Survey (SF-36), the World Health Organization Quality of Life 100 Brief (WHOQOL-BREF), and the Addison's Disease-Specific Quality of Life Questionnaire (AddiQOL). Skeletal and body composition will be assessed by DEXA and biomarkers of bone turnover. Hirsutism in women will be assessed by the modified Ferriman-Gallwey scale and / or self-reported frequency of hair loss in different areas of the body. In women, ovulation cycles will be monitored. In men, testosterone (adjusted for sex hormone-binding globulin) levels will be measured, the presence of TART will be monitored by ultrasound, and testicular function will be assessed by sperm count, motility, and morphology.

[0129] Tapering of hydrocortisone and mineralocorticoid therapy Tapering of hydrocortisone treatment may be initiated at any time following treatment with BBP-631, at the investigator's discretion and if supported by available clinical and laboratory data.

[0130] The following tapering sequence is used as general guidance for hydrocortisone dosing adjustments according to the participant's HPA axis hormone levels: (1) Reduce hydrocortisone dose by up to 25% (to a minimum total dose of 15 mg daily) if 17-OHP level is ≤1200 ng / dL or androstenedione (A4) level is ≤normal; (2) Discontinue hydrocortisone treatment (or consider reducing total daily dose to <15 mg) if ACTH-stimulated cortisol is >14 μg / dL by immunoassay. Otherwise, hydrocortisone treatment is maintained at a minimum dose of 15 mg / day, with no further reductions unless ACTH-stimulated cortisol is >14 μg / dL by immunoassay.

[0131] To ensure participants are tolerating the tapering (or discontinuation) of hydrocortisone: morning laboratory values ​​of 17-OHP, androstenedione (A4), cortisol, and ACTH, and optionally 11-ketotestosterone, will be obtained within 2 weeks of initiating tapering. Follow-up contacts from the site to participants to inquire about clinical status and tolerability of tapering will be added weekly for at least 2 weeks after initiating tapering.

[0132] Closely monitor for signs and symptoms of adrenal insufficiency. Based on the participant's laboratory test trends and / or clinical status, determine whether to increase or restart hydrocortisone dosing if signs and symptoms become clinically evident.

[0133] For participants taking mineralocorticoids, tapering will be permitted at the investigator's discretion only after the Week 6 assessment. The following tapering sequence will be used as general guidance for adjusting mineralocorticoid treatment dosage at the investigator's discretion depending on the participant's hormone levels and clinical symptoms: (1) If plasma renin activity < normal, reduce mineralocorticoid treatment by 50%; (2) If signs or symptoms of mineralocorticoid excess (e.g., marked elevation of blood pressure), consult to reduce mineralocorticoid treatment; and consider discontinuing mineralocorticoid treatment only if peak plasma aldosterone concentrations are ≥ 9 ng / dL during ACTH stimulation.

[0134] As mineralocorticoid dosing is tapered, closely monitor for signs and symptoms of mineralocorticoid deficiency. If signs or symptoms of adrenal insufficiency become clinically evident during the study, mineralocorticoid therapy may be increased or resumed.

[0135] Long-term follow-up period Upon completion of this study, and after providing informed consent, participants will begin a separate long-term follow-up study and will be monitored at appropriate intervals for an additional four years to continue to evaluate the safety and efficacy of BBP-631 treatment. In total, all participants will be followed for at least five years from the date of treatment with BBP-631.

[0136] Dose Escalation and Enrollment Expansion Scheme All dose levels will be investigated based on an escalation and expansion scheme determined by the safety and tolerability of BBP-631. Participants will not be treated at doses higher than dose level 3. See Figure 3.

[0137] During the dose escalation phase, at least 3 (maximum 6) participants from Cohort 1 (i.e., classical CAH and hydrocortisone dose ≤ 30 mg / day) will be enrolled at each dose level, resulting in a total of at least 9 (maximum 18) participants from Cohort 1 receiving BBP-631. Dose escalation will continue based on tolerability of BBP-631. Dose escalation will be determined by tolerability and available data from Cohort 1 participants regarding the effects of BBP-631 on HPA axis hormones.

[0138] Sentinel participants at each BBP-631 dose level are participants with a non-null CYP21A2 mutation in at least one allele, thus reducing the possibility of confounding signals that may be detected from cross-reactivity of immunological agents. Non-sentinel participants at each dose level are enrolled without genotype exclusion. After the first participant receives BBP-631 at the starting dose (dose level 1), treatment of the second participant will occur only after reviewing the safety data of the first participant through at least the 28th day post-dose outpatient visit. As the study progresses, treatment of subsequent participants will be permitted after reviewing the cumulative safety data of all participants through at least the 28th day post-dose outpatient visit.

[0139] Prior to escalation and prior to dosing the second participant in a dose cohort, a review of cumulative post-dose safety data for all participants will be required for at least 4 weeks. Further dose escalation will follow the same algorithm, proceeding only up to dose level 3.

[0140] Safety Monitoring Immunogenicity monitoring and reactive immunomodulation programs

[0141] Each participant will be monitored post-treatment for antibody development and T cell reactivity to the capsid and transgene products weekly from the time of BBP-631 administration until completion of the week 5 safety assessment, and then every three weeks until the end of the study.

[0142] The prophylactic tacrolimus dosing regimen will be maintained for 4 weeks, followed by a tapering period over approximately 2 weeks. If at any time during the study, transaminase levels increase to more than twice the upper limit of normal (ULN) or to a level that is clinically relevant in the investigator's opinion, tacrolimus dosing will be resumed (targeting trough concentrations) or increased. In addition, high doses of prednisone (or hydrocortisone at the investigator's discretion) will be initiated and then rapidly tapered over 3 weeks to baseline glucocorticoid dose levels once transaminase levels have normalized. Concurrently, liver function tests (LFTs) will be monitored weekly or more frequently until LFT levels return to below the ULN. During this monitoring period, tacrolimus and prednisone doses may be adjusted depending on observed LFT levels. If participants discontinue glucocorticoids for 4 weeks and LFTs have normalized, tacrolimus tapering may be resumed. If necessary, a treatment regimen involving increasing the target trough concentration of tacrolimus or administration of other immunomodulatory agents may be initiated.

[0143] Other considerations Any hepatotoxic substances (e.g., ketamine, halogenated anesthetics) are prohibited for at least 12 weeks after BBP-631 administration. Participants are advised to avoid taking acetaminophen (and other similar drugs) during the same period. Participants should avoid substances that inhibit or induce the metabolism of tacrolimus via the CYP3A or CYP3A4 pathways, including grapefruit juice.

[0144] A baseline (5-day evaluation period, such as the 5-day hospital stay) will be performed with detailed assessment of diurnal hormone profile (including 17-OHP and A4), cortisol clearance, ACTH stimulation test, renin and aldosterone, and other investigational hormones.

[0145] The main eligibility criteria for the patient population include: Patients have been diagnosed with CAH: Group 1: diagnosis of classic CAH (simple virilizing or salt-wasting) from childhood based on clinical and genetic evidence, including well-documented CAH history data (i.e., medical history, longitudinal laboratory values, and medication history); or Group 2: diagnosis of classic salt-wasting CAH from childhood based on clinical and genetic evidence, including well-documented CAH history data (i.e., medical history, longitudinal laboratory values, and medication history). Patients meet the 17-OHP level thresholds at the first screening clinic visit: Group 1: 17-OHP level ≥ 10 x ULN (upper limit of normal), but less than 40 x ULN, or Group 2: 17-OHP level ≥ 5 x ULN, but less than 40 x ULN. Patients have androstenedione (A4) levels above the ULN. Patients are receiving a stable regimen of oral hydrocortisone as their sole glucocorticoid maintenance therapy at the following total daily doses: Group 1: ≦30 mg / day hydrocortisone for the treatment of CAH for the 3 months prior to screening, or Group 2: >30 mg / day hydrocortisone for the treatment of CAH for the 3 months prior to screening. If the patient is already receiving mineralocorticoid agonist therapy, the patient has a documented history of needing mineralocorticoid agonist therapy (e.g., fludrocortisone) and having taken a stable dose of this mineralocorticoid agonist for the 3 months prior to screening.

[0146] Dosage The study will use three dose levels of BBP-631: (i) dose level 1: 1.5 x 1013 vg / kg body weight; (ii) dose level 2: 3.0 x 1013 vg / kg body weight; and (iii) dose level 3: 6.0 x 1013 vg / kg body weight. Each treatment-eligible participant will receive a single dose of BBP-631 via IV infusion on day 0 of the study. The duration of the infusion will vary depending on the treatment dose, which is calculated according to the participant's body weight.

[0147] Safety and Efficacy Analysis The optimal dose is selected based on: (i) adverse events (AEs), clinical laboratory measurements (chemistry, hematology, urinalysis); (ii) levels of endogenous cortisol (measured before and after ACTH stimulation, both by mass spectrometry and immunoassays), 17-OHP, A4, and other hormones related to the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes; (iii) renin and aldosterone levels; (iv) changes in hydrocortisone (HC) and mineralocorticoid (MC) medication use; and (v) quality of life assessments measuring the physical and physiological impacts of hormonal imbalance.

[0148] Clinically significant changes in physical examination findings will be reported as AEs. Laboratory evaluations (clinical chemistry, coagulation, hematology, and urinalysis) will be collected to evaluate the safety and tolerability of BBP-631 in adult participants with classical CAH. Laboratory tests and vital signs, 12-lead ECG, and physical examination findings will be collected. Safety and tolerability assessments, and hormonal and other efficacy assessments will be performed to select the optimal dose or dose range of BBP-631 for future studies.

[0149] The following safety and efficacy assessments will be evaluated as listed below.

[0150] [Table 2-1] [Table 2-2] [Table 2-3] Numbered embodiments

[0151] Embodiment 1. A method comprising administering to a subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, The rAAV vector: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; The therapeutically effective amount is about 10 12 vg / kg~about 10 14 The method according to claim 1, wherein the concentration of the active ingredient is in the range of vg / kg.

[0152] Embodiment 2. The method of embodiment 1, wherein the subject is in need of expression of 21OH.

[0153] Embodiment 3. The method of embodiment 1 or 2, wherein the subject has 21OH deficiency (21OHD).

[0154] Embodiment 4. The method of embodiment 3, wherein the subject has 21OHD Prader type IV or V.

[0155] Embodiment 5. The method of embodiment 3 or 4, wherein the subject has congenital adrenal hyperplasia (CAH).

[0156] Embodiment 6. The method of embodiment 5, wherein the CAH is classical CAH.

[0157] Embodiment 7. The method of any one of embodiments 1 to 6, wherein administration of the rAAV vector results in expression of 21OH in the subject.

[0158] Embodiment 8. The method of embodiment 7, wherein said 21OH is expressed in the adrenal cortex, adrenal medulla, adrenal stem cells, adrenal progenitor cells, liver, or ovaries of said subject.

[0159] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the subject is in need of cortisol and / or aldosterone.

[0160] Embodiment 10. The method of any one of embodiments 1-9, wherein the subject has a cortisol deficiency and / or an aldosterone deficiency.

[0161] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the subject has an excess of progesterone, 17-OHP, renin, and / or androstenedione (A4).

[0162] Embodiment 12. The method of embodiment 11, wherein the subject has excess progesterone, 17-OHP, renin, and / or androstenedione (A4) in the blood and / or urine.

[0163] Embodiment 13. A method for expressing 21-hydroxylase (21OH) in a subject in need of such expression, comprising: administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, the rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; The therapeutically effective amount is about 10 12 vg / kg to about 10 14 vg / kg, thereby expressing 21OH in the subject.

[0164] Embodiment 14 The method of embodiment 13, wherein said 21OH is expressed in the adrenal cortex, adrenal medulla, adrenal stem cells, adrenal progenitor cells, liver, or ovaries of said subject.

[0165] Embodiment 15. A method of treating a subject having 21-hydroxylase deficiency (21OHD), comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; and the therapeutically effective amount is about 10 12 vg / kg~about 10 14 vg / kg, thereby treating 21OHD in the subject.

[0166] Embodiment 16. A method of increasing cortisol and / or aldosterone levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector is: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; and the therapeutically effective amount is about 10 12 vg / kg~about 10 14 vg / kg range, thereby increasing the levels of cortisol and / or aldosterone in the subject.

[0167] Embodiment 17. A method of reducing progesterone, 17-OHP, renin, and / or androstenedione (A4) levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector is: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; and the therapeutically effective amount is about 10 12 vg / kg~about 10 14vg / kg, thereby lowering the levels of progesterone, 17-OHP, renin, and / or androstenedione (A4) in the subject.

[0168] Embodiment 18. The method of any one of embodiments 13 to 17, wherein the subject has Prader type IV or V 21-hydroxylase deficiency (21OHD).

[0169] Embodiment 19. The method of any one of embodiments 13-17, wherein the subject has congenital adrenal hyperplasia (CAH).

[0170] Embodiment 20. The method of embodiment 19, wherein the CAH is classical CAH.

[0171] Embodiment 21. The therapeutically effective amount is about 10 13 vg / kg~about 10 14 The method of any one of embodiments 1 to 20, wherein the IgG4-dependent agonist activity is in the range of vg / kg.

[0172] Embodiment 22. The therapeutically effective amount is about 1.5×10 13 21. The method of any one of embodiments 1-20, wherein the dose is 100 mg / kg.

[0173] Embodiment 23. The therapeutically effective amount is about 3×10 13 21. The method of any one of embodiments 1-20, wherein the dose is 100 mg / kg.

[0174] Embodiment 24. The therapeutically effective amount is about 6×10 13 21. The method of any one of embodiments 1-20, wherein the dose is 100 mg / kg.

[0175] Embodiment 25. The method of any one of embodiments 1 to 24, further comprising selecting a subject having 21-hydroxylase deficiency (21OHD) prior to the administering step.

[0176] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the rAAV vector is administered to the subject intravenously by direct injection into the adrenal gland via laparotomy or laparoscopy, or by injection into the adrenal artery or vein via catheter insertion.

[0177] Embodiment 27 The method of embodiment 26, wherein said direct injection into the adrenal gland is direct injection into the adrenal cortex.

[0178] Embodiment 28. The method of any one of embodiments 1 to 25, wherein the rAAV vector is administered by intravenous (IV) infusion.

[0179] Embodiment 29. The method of any one of embodiments 1 to 28, wherein the 21OH protein is a human 21OH protein.

[0180] Embodiment 30. The method of any one of embodiments 1 to 29, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of a cDNA for human 21OH (CYP21A2).

[0181] Embodiment 31. The method of any one of embodiments 1 to 30, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of a codon-optimized nucleotide sequence.

[0182] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of SEQ ID NO:50.

[0183] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the non-AAV nucleotide sequence encoding a 21OH protein encodes an amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1.

[0184] Embodiment 34 The method of any one of embodiments 1 to 33, wherein the promoter directs expression of the 21OH protein in a host cell.

[0185] Embodiment 35 The method of embodiment 34, wherein the host cell is an adrenal cell or an adrenal cortical cell.

[0186] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the promoter is a cytomegalovirus / β-actin hybrid promoter, a PGK promoter, or a promoter specific for expression in adrenal cortical cells.

[0187] Embodiment 37. The method of embodiment 36, wherein the cytomegalovirus / β-actin hybrid promoter is a CAG, CB6, or CBA promoter.

[0188] Embodiment 38. The method of any one of embodiments 1 to 37, wherein the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49.

[0189] Embodiment 39. The method of any one of embodiments 1 to 38, wherein the ITR is an ITR of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype.

[0190] Embodiment 40. The method of any one of embodiments 1 to 39, wherein the rAAV is of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype.

[0191] Embodiment 41 The method of any one of embodiments 1 to 39, wherein the rAAV is an AAV5 serotype.

[0192] Embodiment 42. The method of any one of embodiments 1 to 41, wherein the nucleic acid molecule further comprises a Kozak sequence.

[0193] Embodiment 43 The method of any one of embodiments 1 to 42, wherein the nucleic acid molecule further comprises a miR-122 binding site.

[0194] Embodiment 44. About 10 12 vg / kg~about 10 14 1. A method comprising administering to a subject vg / kg of a recombinant adeno-associated virus 5 (rAAV5) vector, wherein the rAAV5 vector comprises: a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, wherein the non-AAV nucleotide sequence is operably linked to a promoter that directs expression of the 21OH protein in an adrenal or adrenal cortical cell.

[0195] Embodiment 45. About 10 13 vg / kg~about 10 14 The method of embodiment 44, wherein vg / kg of the rAAV5 vector is administered to the subject.

[0196] Embodiment 46. Approximately 1.5 x 10 13 The method of embodiment 45, wherein vg / kg of the rAAV5 vector is administered to the subject.

[0197] Embodiment 47. Approximately 3 x 10 13 The method of embodiment 45, wherein vg / kg of the rAAV5 vector is administered to the subject.

[0198] Embodiment 48. Approximately 6 x 10 13 The method of embodiment 45, wherein vg / kg of the rAAV5 vector is administered to the subject.

[0199] Embodiment 49. The method of any one of embodiments 45 to 48, wherein the rAAV5 vector is administered intravenously to the subject.

[0200] Embodiment 50. The method of any one of embodiments 44-49, wherein the subject has 21-hydroxylase deficiency (21OHD).

[0201] Embodiment 51. A method of treating a subject having 21-hydroxylase deficiency (21OHD), comprising administering to a subject about 1.5×10 13 The method comprises intravenously administering to the subject vg / kg of a recombinant adeno-associated virus 5 (rAAV5) vector, wherein the rAAV5 vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, the non-AAV nucleotide sequence being operably linked to a promoter that directs expression of the 21OH protein in adrenal or adrenal cortical cells, thereby treating 21OHD in the subject.

[0202] Embodiment 52. A method of treating a subject having 21-hydroxylase deficiency (21OHD), comprising administering to a subject about 3×10 13 The method comprises intravenously administering to the subject vg / kg of a recombinant adeno-associated virus 5 (rAAV5) vector, wherein the rAAV5 vector comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, the non-AAV nucleotide sequence being operably linked to a promoter that directs expression of the 21OH protein in adrenal or adrenal cortical cells, thereby treating 21OHD in the subject.

[0203] Embodiment 53. A method of treating a subject having 21-hydroxylase deficiency (21OHD), comprising administering to a subject about 6×10 13 Administering intravenously to the subject a recombinant adeno-associated virus 5 (rAAV5) vector at 100 mg / kg, wherein the rAAV5 vector is: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, wherein the non-AAV nucleotide sequence is operably linked to a promoter that directs expression of the 21OH protein in an adrenal cell or an adrenal cortical cell, thereby treating 21OHD in the subject.

[0204] Embodiment 54. The method of any one of embodiments 44 to 53, wherein the promoter is a cytomegalovirus / β-actin hybrid promoter, a PGK promoter, or a promoter specific for expression in adrenal cortical cells.

[0205] Embodiment 55. The method of embodiment 54, wherein the cytomegalovirus / β-actin hybrid promoter is a CAG, CB6, or CBA promoter.

[0206] Embodiment 56. The method of any one of embodiments 44 to 55, wherein the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, or SEQ ID NO:49.

[0207] Embodiment 57. The method of any one of embodiments 44 to 56, wherein the ITR is an ITR of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, or rh74 serotype.

[0208] Embodiment 58. The method of any one of embodiments 44 to 57, wherein the nucleic acid molecule further comprises a Kozak sequence.

[0209] Embodiment 59. The method of any one of embodiments 44 to 58, wherein the nucleic acid molecule further comprises a miR-122 binding site.

[0210] Embodiment 60. The method of any one of embodiments 44 to 59, wherein the subject has Prader type IV or V 21-hydroxylase deficiency (21OHD).

[0211] Embodiment 61. The method of any one of embodiments 44-59, wherein the subject has congenital adrenal hyperplasia (CAH).

[0212] Embodiment 62. The method of embodiment 61, wherein the CAH is classical CAH.

[0213] Embodiment 63. The method of any one of embodiments 1 to 62, further comprising administering to the subject a therapeutically effective amount of an immunosuppressant.

[0214] Embodiment 64. A method comprising administering to a subject a therapeutically effective amount of a recombinant adeno-associated virus 5 (rAAV5) vector and a therapeutically effective amount of an immunosuppressant, wherein the rAAV5 comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, wherein the non-AAV nucleotide sequence is operably linked to a promoter directing expression of the 21OH protein in an adrenal or adrenal cortical cell.

[0215] Embodiment 65. The method of embodiment 64, wherein the subject has 21-hydroxylase deficiency (21OHD).

[0216] Embodiment 66. A method of treating a subject having 21-hydroxylase deficiency (21OHD), comprising intravenously administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus 5 (rAAV5) vector and a therapeutically effective amount of an immunosuppressant, wherein the rAAV5 comprises a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR) and a non-AAV nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, wherein the non-AAV nucleotide sequence is operably linked to a promoter that directs expression of the 21OH protein in an adrenal or adrenal cortical cell, thereby treating 21OHD in the subject.

[0217] Embodiment 67. The method of any one of embodiments 63 to 66, wherein the immunosuppressant is administered prior to, simultaneously with, and / or after administration of the rAAV vector.

[0218] Embodiment 68. The method of embodiment 67, wherein the immunosuppressant is administered prior to the administration of the rAAV vector.

[0219] Embodiment 69. The method of embodiment 68, wherein the immunosuppressant is administered at least 12 hours prior to the administration of the rAAV vector.

[0220] Embodiment 70. The method of embodiment 69, wherein the immunosuppressant is administered about 2 days prior to the administration of the rAAV vector.

[0221] Embodiment 71. The method of any one of embodiments 63 to 70, wherein the immunosuppressant is a non-glucocorticoid immunosuppressant.

[0222] Embodiment 72. The method of any one of embodiments 63 to 70, wherein the immunosuppressant is an inhibitor of calcineurin.

[0223] Embodiment 73. The method of any one of embodiments 63 to 72, wherein the immunosuppressant is cyclosporine, tacrolimus, sirolimus, everolimus, zotarolimus, or any combination thereof.

[0224] Embodiment 74. The method of embodiment 73, wherein the immunosuppressant is tacrolimus.

[0225] Embodiment 75. The method of any one of embodiments 63-74, wherein the immunosuppressant is administered orally.

[0226] Embodiment 76. The method of any one of embodiments 63-75, wherein the therapeutically effective amount of the immunosuppressant is in the range of about 0.01 mg / kg to about 0.05 mg / kg.

[0227] Embodiment 77. The method of embodiment 76, wherein said therapeutically effective amount of said immunosuppressant is about 0.025 mg / kg.

[0228] Embodiment 78. The method of any one of embodiments 63-77, wherein the therapeutically effective amount of the immunosuppressant is administered twice daily.

[0229] Embodiment 79. The method of any one of embodiments 1 to 78, comprising administering to the subject the therapeutically effective amount of a steroid.

[0230] Embodiment 80. The method of embodiment 79, wherein the steroid is a mineralocorticoid.

[0231] Embodiment 81. The method of embodiment 79, wherein the steroid is a glucocorticoid.

[0232] Embodiment 82. The method of embodiment 79, wherein the steroid is hydrocortisone.

[0233] Embodiment 83. The method of any one of embodiments 79 to 82, wherein the steroid is administered prior to, simultaneously with, and / or after administration of the rAAV vector.

[0234] Embodiment 84. The method of embodiment 83, wherein the therapeutically effective amount of the steroid administered to the subject prior to administration of the rAAV vector is greater than the therapeutically effective amount of the steroid administered to the subject after administration of the rAAV vector.

[0235] Embodiment 85. The method of any one of embodiments 1 to 84, wherein the subject has higher levels of cortisol and / or aldosterone after administration of the rAAV compared to before administration of the rAAV.

[0236] Embodiment 86. The method of any one of embodiments 1 to 85, wherein the subject has higher levels of cortisol and / or aldosterone after administration of the rAAV compared to a control subject having 21-hydroxylase deficiency (21OHD) who is not administered the rAAV.

[0237] Embodiment 87. The method of any one of embodiments 1 to 86, wherein the subject has lower levels of progesterone, 17-OHP, renin and / or androstenedione (A4) after administration of the rAAV compared to before administration of the rAAV.

[0238] Embodiment 88. The method of any one of embodiments 1 to 87, wherein the subject has lower levels of progesterone, 17-OHP, renin and / or androstenedione (A4) after administration of the rAAV compared to a control subject having 21-hydroxylase deficiency (21OHD) who is not administered the rAAV.

[0239] Embodiment 89. The method of any one of embodiments 1 to 88, wherein the rAAV vector comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO:52.

Claims

1. A pharmaceutical composition for the treatment of 21OH deficiency (21OHD), said pharmaceutical composition comprising a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) vector, said rAAV vector comprising: (i) a nucleic acid molecule comprising at least one AAV inverted terminal repeat (ITR); (ii) a non-AAV nucleotide sequence encoding a 21-hydroxylase (21OH) protein, wherein the non-AAV nucleotide sequence is operably linked to a promoter; The therapeutically effective amount is about 10 １２ Vector genomes / kilogram (vg / kg) ~ about 10 １４ The pharmaceutical composition wherein the total amount of the pharmaceutical composition is in the range of vg / kg.

2. The pharmaceutical composition of claim 1, wherein the 21OHD is Prader classification IV or V.

3. The pharmaceutical composition according to claim 1 or 2, wherein the 21OHD is congenital adrenal hyperplasia (CAH).

4. The pharmaceutical composition of claim 3, wherein the CAH is classical CAH.

5. The pharmaceutical composition of any one of claims 1 to 4, configured to induce expression of 21OH in the adrenal cortex, adrenal medulla, adrenal stem cells, adrenal progenitor cells, liver, or ovaries of a subject upon administration of the pharmaceutical composition to the subject.

6. The therapeutically effective amount is about 10 １３ vg / kg ~ approx. 10 １４ The pharmaceutical composition according to any one of claims 1 to 5, wherein the total amount of the compound is in the range of vg / kg.

7. The therapeutically effective amount is about 1.5×10 １３ The pharmaceutical composition according to any one of claims 1 to 6, wherein the total amount of the pharmaceutical composition is 100 mg / kg.

8. The therapeutically effective amount is about 3×10 １３ The pharmaceutical composition according to any one of claims 1 to 6, wherein the total amount of the pharmaceutical composition is 100 mg / kg.

9. The therapeutically effective amount is about 6×10 １３ The pharmaceutical composition according to any one of claims 1 to 6, wherein the total amount of the pharmaceutical composition is 100 mg / kg.

10. The pharmaceutical composition of any one of claims 1 to 9, which is configured to be administered intravenously to a subject by direct injection into the adrenal gland via open surgery or laparoscopy, or by injection into the adrenal artery or vein via catheter insertion.

11. 11. The pharmaceutical composition of claim 10, adapted to be administered to a subject by direct injection into the adrenal cortex.

12. The pharmaceutical composition of any one of claims 1 to 11, configured to be administered to a subject by intravenous (IV) injection.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the 21OH protein is a human 21OH protein.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of the cDNA for human 21OH (CYP21A2).

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of a codon-optimized nucleotide sequence.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the non-AAV nucleotide sequence encoding a 21OH protein comprises or consists of SEQ ID NO:

50.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the non-AAV nucleotide sequence encoding a 21OH protein encodes an amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

1.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the promoter is configured to direct expression of the 21OH protein in a host cell.

19. 19. The pharmaceutical composition of claim 18, wherein the host cell is an adrenal cell or an adrenal cortical cell.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the promoter is a cytomegalovirus / β-actin hybrid promoter, a PGK promoter, or a promoter specific for expression in an adrenal cortical cell.

21. 21. The pharmaceutical composition of claim 20, wherein the cytomegalovirus / β-actin hybrid promoter is a CAG, CB6, or CBA promoter.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48 or SEQ ID NO:

49.

23. 23. The pharmaceutical composition of any one of claims 1 to 22, wherein the ITR is an ITR of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, or rh74 serotype.

24. 24. The pharmaceutical composition of any one of claims 1 to 23, wherein the rAAV is of the serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, or rh74.

25. The pharmaceutical composition of any one of claims 1 to 24, wherein the rAAV is an AAV5 serotype.

26. The pharmaceutical composition of any one of claims 1 to 25, wherein the nucleic acid molecule further comprises a Kozak sequence.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the nucleic acid molecule further comprises a miR-122 binding site.

28. A pharmaceutical composition described in any one of claims 1 to 27, configured to be administered in combination with a therapeutically effective amount of an immunosuppressant.

29. 29. The pharmaceutical composition of claim 28, configured to be administered prior to, simultaneously with, and / or after the immunosuppressant agent.

30. 30. The pharmaceutical composition of claim 28 or 29, wherein the immunosuppressant is a non-glucocorticoid immunosuppressant.

31. The pharmaceutical composition according to any one of claims 28 to 30, wherein the immunosuppressant is a calcineurin inhibitor.

32. The pharmaceutical composition of any one of claims 28 to 31, wherein the immunosuppressant is cyclosporine, tacrolimus, sirolimus, everolimus, zotarolimus, or any combination thereof.

33. 33. The pharmaceutical composition of any one of claims 28-32, wherein said therapeutically effective amount of said immunosuppressant is in the range of about 0.01 milligram per kilogram (mg / kg) to about 0.05 mg / kg.

34. A pharmaceutical composition described in any one of claims 1 to 33, configured to be administered in combination with a therapeutically effective amount of a steroid.

35. 35. The pharmaceutical composition of claim 34, wherein the steroid is a mineralocorticoid, a glucocorticoid or a hydrocortisone.

36. 36. The pharmaceutical composition of claim 34 or 35, adapted to be administered prior to, simultaneously with, and / or after administration of the steroid.

37. The pharmaceutical composition of any one of claims 1 to 36, wherein the rAAV vector comprises an expression cassette comprising the nucleic acid sequence of SEQ ID NO:52.

Images