Recombinant adeno-associated virus vector for the treatment of crystalline retinal degeneration

Abstract

Provided are a recombinant adeno-associated vector and use of the recombinant adeno-associated vector for treating crystalline retinal degeneration (BCD). The recombinant adeno-associated vector comprises a codon-optimized sequence encoding CYP4V2 linked to a selected gene expression regulatory sequence.

Description

Technical Field

[0001] The present disclosure provides expression cassettes and recombinant adeno-associated virus (rAAV) vectors comprising nucleic acids encoding CYP4V2. Viral particles comprising rAAV vectors, compositions comprising viral particles, and uses thereof are also provided. Background Art

[0002] Recent advances have shown that gene therapy holds great promise for treating human genetic disorders [1]. More than 6,000 genetic diseases have been described [2], and the number of genetic diseases is still increasing as genome sequencing technology continues to reveal [3]. In recent years, the discovery of safer delivery vectors and the unveiling of new gene editing technologies have significantly enhanced the toolbox for treating these genetic diseases, whereby the defective gene can be supplemented via exogenous DNA carried by therapeutic vectors or permanently corrected in situ via gene editing [4]. In contrast to this promising prospect, the list of clinically treatable genetic disorders has grown slowly [5]. A major limitation of this situation is the lack of animal models that are translatable to humans, which has hampered the evaluation of the therapeutic effects of gene therapy [6].

[0003] Bietti Crystalline Dystrophy (BCD, MIM 210370) is an autosomal recessive genetic disease first described by Italian ophthalmologist GB Bietti in 1937. It was named after the obvious yellow-white crystalline deposits observed in the fundus of patients[7]. In Europe, BCD accounts for approximately 3% of all non-syndromic retinitis pigmentosa (RP) and approximately 10% of non-syndromic autosomal recessive RP[8]. BCD also appears to be more common in East Asia, especially in China, with a gene mutation frequency estimated to be 1 / 20,000[9].

[0004] In 2000, the genetic basis of BCD was identified as being linked to chromosome 4q35 [8]. CYP4V2, a member of the cytochrome P450 superfamily, was identified as the causative gene for BCD in 2004

[10] . In addition to crystalline deposits in the retina and cornea, altered fatty acid concentrations have been found in the serum of BCD patients, implying dysregulation of lipid metabolism [11,12]. More importantly, BCD patients inevitably develop vision loss and night blindness between the ages of 20 and 40, and eventually become legally blind between the ages of 50 and 60

[11] . Unfortunately, 80 years after BCD was first discovered, there is still no treatment for this severe blinding disease.

[0005] AAV has become the delivery vehicle of choice for gene therapy to treat genetic disorders such as BCD due to its demonstrated long-term transgene expression, selective tropism, and non-pathogenic properties.

[0006] CN111733174B discloses a construct for treating BCD, which can be packaged into an AAV vector and comprises the coding sequences of both CYP4V2 and RdCVF. The construct comprises a CAG promoter and a BGH polyadenylation signal site.

[0007] CN109136266A also provides a construct for expressing CYP4V2 by rAAV. A sequence encoding a short peptide C9 is inserted into the packaging plasmid to enable it to specifically bind to the CD59 antigen on the human RPE cell membrane. In addition, a sequence encoding the HRH peptide is inserted together with the coding sequence of CYP4V2 to inhibit VEGF.

[0008] Both CN111733174B and CN109136266A used the wild-type coding sequence of CYP4V2.

[0009] Therefore, there is a need in the art for improved gene therapy compositions and methods that can effectively and safely restore CYP4V2 gene function in BCD patients. Summary of the invention

[0010] The inventors developed an rAAV vector comprising a codon-optimized coding sequence of CYP4V2 and a combination of certain gene expression regulatory sequences, which achieved significantly enhanced expression levels (about 26.1 times) compared to the previously published form

[13] , thereby completing the present invention.

[0011] Thus, in a first aspect, the present disclosure relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NOs: 2-17 and encoding human CYP4V2. In a more preferred embodiment, the nucleotide sequence is selected from SEQ ID NOs: 8, 9, 15, 16 and 17. In an even more preferred embodiment, the nucleotide sequence is SEQ ID NO: 8 or SEQ ID NO: 16. In a most preferred embodiment, the nucleotide sequence is SEQ ID NO: 16.

[0012] In an embodiment of the first aspect, the isolated nucleic acid molecule further comprises a promoter, the promoter being operably linked to the 5' of the nucleotide sequence encoding CYP4V2. Preferably, the promoter is the CAG promoter of SEQ ID NO:35.

[0013] In one embodiment, the isolated nucleic acid molecule further comprises a polyadenylation sequence 3' to the nucleotide sequence encoding the CYP4V2 polypeptide. Preferably, the polyadenylation sequence is bovine growth hormone (bGH) poly A, synthetic poly A (SPA) or simian virus 40 (SV40) poly A, more preferably SV40 poly A.

[0014] In another embodiment, the isolated nucleic acid molecule further comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) located between the CYP4V2 coding sequence and the SV40 poly A sequence.

[0015] In a preferred embodiment, the isolated nucleic acid molecule comprises

[0016] (a) a nucleotide sequence of any one of SEQ ID NOs: 18-34, or

[0017] (b) a nucleotide sequence having at least 85% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology to any one of SEQ ID NOs: 18-34.

[0018] In a second aspect, the present disclosure provides a recombinant AAV (rAAV) vector comprising the nucleic acid molecule of the first aspect.

[0019] In one embodiment, the rAAV vector comprises at least one ITR, preferably two ITRs. In a preferred embodiment, the two ITRs are derived from AAV2 ITRs.

[0020] In a third aspect, the disclosure provides a viral particle comprising the recombinant AAV vector of the second aspect and an AAV capsid selected from AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8, AAV Anc80L65 and variants thereof. Preferably, the capsid is AAV8.

[0021] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising the viral particle of the third aspect and a pharmaceutically acceptable excipient.

[0022] In a fifth aspect, the present disclosure provides use of the rAAV vector in the manufacture of a medicament for treating or preventing crystalline retinal degeneration (BCD) or any other disease of retinal pigment epithelium (RPE) atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1is an illustrative diagram showing the constructs CMV-hCyp4v2 (upper panel) and CAG-hCyp4v2 (BCD1, lower panel).

[0024] Figure 2 is a histogram showing the CYP4V2 transgenic protein expression levels in HEK293 of the construct CMV-hCyp4v2 compared to the construct CAG-hCyp4v2(BCD1).

[0025] Figure 3 Evaluation of CYP4V2 transgenic protein expression levels of constructs BCD1-BCD17 in HEK293 cells is shown.

[0026] Figure 4 Evaluation of CYP4V2 transgenic protein expression levels of construct BCD1-BCD17 in ARPE-19 cells is shown.

[0027] Figure 5 A- Figure 5 B shows the CYP4V2 transgenic protein expression levels of constructs BCD8, BCD9, BCD15, BCD16 and BCD17 in HEK293 cells by Western blot (A) and histogram (B).

[0028] Figure 6 A- Figure 6 B shows the CYP4V2 transgenic protein expression levels of constructs BCD8, BCD9, BCD15, BCD16 and BCD17 in ARPE-19 cells by Western blot (A) and histogram (B).

[0029] Figure 7 A- Figure 7 B shows the CYP4V2 transgene protein expression levels of packaged AAV8-BCD1, AAV8-BCD8, and AAV8-BCD16 in transduced ARPE-19 cells by Western blot (A) and histogram (B). DETAILED DESCRIPTION

[0030] Unless specifically defined elsewhere in this document, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0032] In the context of the present disclosure, unless otherwise indicated, the word "comprise" and its variations such as "comprises" and "comprising" will be understood to imply the inclusion of the stated elements (e.g., amino acid sequences, nucleotide sequences, characteristics, steps or groups thereof), but not the exclusion of any other elements (e.g., amino acid sequences, nucleotide sequences, characteristics and steps). When used herein, the term "comprises" or any variation thereof may be replaced by the terms "containing", "including" or sometimes by "having" or its equivalents. In certain embodiments, the word "comprising" also includes the case of "consisting of".

[0033] The abbreviation "CYP4V2" refers to cytochrome P450 family 4 subfamily V member 2, which is a CYP4V2 protein encoding gene in the cytochrome P450 heme thiolate protein superfamily that is involved in various substrates in the oxidative metabolic pathway. It is known that defects in the gene CYP4V2 can cause BCD and also fundus dystrophy. Unless otherwise indicated, CYP4V2 mentioned herein refers to human CYP4V2 in the context of the present disclosure.

[0034] Isolated nucleic acid encoding CYP4V2 protein .

[0035] The present disclosure provides an isolated nucleic acid sequence comprising a nucleotide sequence encoding a CYP4V2 protein (particularly human CYP4V2). "Isolated nucleic acid" means DNA or RNA removed from all or part of a polynucleotide (the isolated polynucleotide is present in the polynucleotide in nature) or DNA or RNA connected to a polynucleotide to which it is not connected in nature. An isolated nucleic acid molecule "comprising" a specific nucleotide sequence may also contain, in addition to the specified sequence, an operably linked regulatory sequence that controls the expression of the coding region of the listed nucleic acid sequence. Due to codon degeneracy, those skilled in the art will appreciate that a specific amino acid sequence may be encoded by different nucleotide sequences.

[0036] The nucleotide sequences encoding CYP4V2 of the present disclosure have been codon-optimized and screened, resulting in enhanced expression levels compared to the wild-type coding sequence that has not been codon-optimized.

[0037] For example, according to Western blot analysis, in HEK293 cells and ARPE-19 cells, the codon-optimized coding sequence of CYP4V2 can achieve about 2 times, about 3 times, about 4 times, about 5 times, about 10 times, about 15 times, or about 20 times the CYP4V2 protein expression level compared to the wild-type coding sequence.

[0038] Preferably, the codon-optimized coding sequence of CYP4V2 has a reduced number of CpGs and / or a reduced number of CpG islands. For example, the codon-optimized coding sequence comprises less than 100 CpGs, less than 70 CpGs, less than 20 CpGs, less than 15 CpGs, less than 10 CpGs, less than 5 CpGs or 0 CpGs. For example, the codon-optimized coding sequence does not have a CpG island. "CpG island" represents a DNA region with a length of at least 200bp, a GC percentage content greater than 50% and an observed CpG to expected CpG ratio greater than 60%.

[0039] For example, compared to the wild-type coding sequence of CYP4V2 (as shown in SEQ ID NO:1), the codon-optimized coding sequence has a sequence identity percentage lower than 80%. The identity percentages of the two sequences can be calculated by programs known in the art. Due to different comparison parameters and different definitions of identity, the calculated identity percentages of the two sequences can vary depending on the computing program used. In the present disclosure, blastn is used to obtain the identity percentages of two nucleotide sequences, and the "megablast" mode is specifically selected.

[0040] The codon-optimized coding sequence may be selected from the nucleotide sequences shown in SEQ ID NOs: 2 to 17. Preferably, the codon-optimized coding sequence is any one of SEQ ID NOs: 8, 9, 15, 16 and 17. More preferably, the codon-optimized coding sequence is SEQ ID NO: 16.

[0041] The regulatory sequence contained in the nucleic acid molecule can be selected from one or more of a promoter, an enhancer, a polyadenylation sequence and a translation termination signal. Certain combinations of regulatory sequences of the present disclosure can achieve unexpected results in improving the expression efficiency of the coding sequence.

[0042] The promoter of the present disclosure can be a constitutive promoter, a tissue-specific promoter or a cell type-specific promoter. For example, the promoter can be an RPE cell-specific promoter. The promoter can be the natural promoter of CYP4V2. In a preferred embodiment, the promoter is a CAG promoter with a sequence as shown in SEQ ID NO.35. The CAG promoter is a strong constitutive promoter that drives high-level gene expression in mammalian expression vectors. The CAG promoter is composed of a cytomegalovirus (CMV) early enhancer element, a promoter selected from the first exon and the first intron of the chicken beta-actin gene, and a splicing acceptor of the rabbit beta-globin gene.

[0043] The Kozak consensus sequence (Kozak sequence), named after the scientist who discovered it, is a nucleic acid motif in most eukaryotic mRNA transcripts that naturally serves as a protein translation start site

[14] . The Kozak sequence ensures correct protein translation and enhances protein expression.

[0044] In addition, the nucleic acid of the present disclosure may further comprise an intron inserted between the promoter and the coding sequence. As known in the art, some introns can enhance the expression of genes in eukaryotic organisms. The intron of the present disclosure may be part of a naturally occurring intron in the CYP4V2 gene.

[0045] The polyadenylation sequence of the present disclosure may be bGH polyA, SPA or SV40 polyA

[15] , preferably SV40 polyA. Any of the polyA may be combined with a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)

[16] , which is a DNA sequence that, when transcribed, produces a tertiary structure that enhances expression and is configured as WPRE-bGH polyA, WPRE-SPA or WPRE-SV40 polyA, respectively.

[0046] In a preferred embodiment of the present disclosure, the nucleic acid sequence comprises a nucleotide sequence encoding: a CAG promoter, a Kozak sequence, a codon-optimized coding sequence of a CYP4V2 gene, and WPRE-SV40 poly A. For example, the isolated nucleic acid sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 18-34. In a more preferred embodiment, the isolated nucleic acid sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 25, 26, 32, 33, and 34. In a most preferred embodiment, the isolated nucleic acid sequence comprises a nucleotide sequence of SEQ ID NO: 33.

[0047] rAAV vectors and viral particles

[0048] The nucleic acid molecules of the present disclosure can be constructed into recombinant AAV vectors to obtain rAAV particles for delivery to a subject for treatment.

[0049] In addition to the inserted nucleotide sequence as described above, the rAAV vector is also in a single-stranded form. The rAAV vector is generally composed of two inverted terminal repeat (ITR) sequences at both ends of the inserted nucleotide sequence. The ITR of the present disclosure may be an ITR derived from an AAV serotype. When referring to the serotype of an AAV ITR, the phrase "derived from" means that the ITR may be an ITR of a certain serotype or a variant with one or more modifications derived therefrom. In a preferred embodiment of the present disclosure, the rAAV vector comprises two ITRs derived from AAV2

[17] . For example, the rAAV vector comprises two AAV2 ITRs, or comprises a wild-type AAV2 ITR and an AAV2 ITR variant lacking region C or region C'. For example, the wild-type AAV2 ITR is located at the 5' position of the inserted nucleotide sequence, while the AAV2 ITR variant is located at the 3' position of the inserted nucleotide sequence; vice versa.

[0050] The rAAV genome is packaged into an AAV capsid. The capsid can be derived from any AAV serotype known in the art or characterized in the future. The capsid and ITR can be derived from the same serotype of AAV or from different serotypes of AAV. Preferably, the capsid is suitable for ocular delivery, such as subretinal, intravitreal or intraocular delivery. In a specific embodiment, the AAV vector comprises a capsid of AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8 or AAVAnc80L65 serotype or a variant thereof.

[0051] In a preferred embodiment of the present disclosure, rAAV is composed of AAV serotype 8 capsid

[18] . There is evidence that ssAAV8 is more effective than ssAAV2 and ssAAV5 in transducing photoreceptors and retinal pigment epithelial cells (RPE). Under the guidance of the capsid protein, the virus transduces the target cells and the transferred genome containing the CYP4V2 gene and regulatory elements is released into the target RPE. In the treated subject, the released rAAV genome remains stable and independent of the host genome to stably produce functional CYP4V2 protein.

[0052] Pharmaceutical composition

[0053] The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical composition of the present disclosure includes an isolated nucleic acid, an rAAV vector or viral particle of the present disclosure, and a pharmaceutically acceptable excipient. Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients.

[0054] Therapeutic Uses

[0055] The rAAV, viral particles or compositions of the present disclosure can be used to treat or prevent BCD associated with CYP4V2 mutations. In addition, the rAAV, viral particles or compositions of the present disclosure can also effectively treat or prevent other conditions or diseases associated with retinal pigment epithelium (RPE) atrophy, such as fundus dystrophy.

[0056] The terms "treat," "treating," or "treatment" include curing or at least alleviating the symptoms of BCD or a condition or disease associated with RPE atrophy.

[0057] Application

[0058] When applied to a subject (e.g., an animal, including a human), or to a cell, tissue, organ, or biological fluid, the terms "administration" and "administering" as used herein mean contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid. The term "administering" also includes in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding compound, or by another cell.

[0059] In the present disclosure, the viral particles or pharmaceutical compositions of the present application are preferably delivered via intraocular delivery, such as subretinal injection.

[0060] Example

[0061] Example 1. Selection of promoter

[0062] The inventors first prepared an AAV vector expressing CYP4V2, which consisted of two ITRs, a CMV promoter, a wild-type CYP4V2 CDS, and SV40 poly A

[13] . Due to the reported risk of silencing of the CMV promoter

[19] , the CMV promoter was later replaced by a CAG promoter, resulting in a new vector named “CAG-hCyp4v2(BCD1)”, which showed significantly improved CYP4V2 protein expression ( Figure 1 , small picture below).

[0063] Example 2. Codon optimization increases Cyp4v2 expression

[0064] In order to obtain a transgenic construct that can provide a higher level of CYP4V2 protein expression, the coding sequence of CYP4V2 was codon optimized to increase the frequency of codon usage in human cells. In addition, the GC content and CpG islands of the modified coding sequence were reduced to minimize the possibility of TLR9-mediated in vivo immune response. Based on the above criteria, 16 different coding sequences (SEQ ID NO: 2-17) were designed and synthesized, and cloned into the CAG promoter to obtain 16 more transgenic constructs (BCD2-BCD17). Table 1 below shows the CpG number, CpG island number and sequence homology of SEQ ID NO: 2-17 with the wild-type sequence.

[0065] Table 1. Comparison of CpG content, number of CpG islands and percentage of sequence identity between 16 codon-optimized sequences and the wild-type coding sequence

[0066] Construct ID Codon ID CpG number CpG islands % identity BCD1 CYP4V2_wt 50 Yes 100 BCD2 CYP4V2_co1 69 Yes 77.15 BCD3 CYP4V2_co2 63 Yes 78.23 BCD4 CYP4V2_co3 62 Yes 76.89 BCD5 CYP4V2_co4 65 YS 77.34 BCD6 CYP4V2_co5 61 Yes 75.56 BCD7 CYP4V2_co6 0 No 79.25 BCD8 CYP4V2_co7 12 No 78.68 BCD9 CYP4V2_co8 0 No 79.12 BCD10 CYP4V2_co9 0 No 78.99 BCD11 CYP4V2_co10 0 No 79.69 BCD12 CYP4V2_co11 0 No 78.29 BCD13 CYP4V2_co12 0 No 78.8 BCD14 CYP4V2_co13 0 No 79.44 BCD15 CYP4V2_co14 0 No 78.74 BCD16 CYP4V2_co15 0 No 78.74 BCD17 CYP4V2_co16 1 No 76.89

[0067] Example 3. Transfection of HEK293 and ARPE-19 cells and detection of expression levels

[0068] HEK293 and ARPE-19 cells were maintained in DMEM + 10% FBS and passaged every 3 days by TrypLE. One day before transfection, HEK293 cells were plated at 1 × 10 5 cells / cm 2 The density of ARPE-19 cells was 7×10 4 cells / cm 2 The density of 24-well plates was inoculated. Lipofectamine 3000 transfection reagent (Invitrogen, L3000008) was used to transfect plasmids according to the user guide. After transfection for 72 hours, cells were collected in RIPA lysis buffer (Beyotime, P0013C) and SDS-PAGE loading buffer (Cowin Bio, CW0027) with a protease inhibitor mixture (Roche, 04693159001), denatured for 15 min at 95 ° C, and centrifuged for 10 min at 12,000 rpm. Supernatant was separated in 4%-10% SDS-PAGE gel (CowinBio, CW0022M), and blotted on 0.45 μm NC transfer membrane (Merck, HATF00010).

[0069] The protein expression levels of CYP4V2 and housekeeping gene GAPDH were detected by antibodies against human CYP4V2 (Sigma, HPA029122) and GAPDH (Abcam, ab8245), respectively. The grayscale value of the band was calculated and normalized to BCD1.

[0070] The results of repeated experiments showed that among the 16 tested codon-optimized vectors, transgenic constructs BCD8, BCD9, BCD15, BCD16, and BCD17 expressed higher levels of CYP4V2 protein, as shown in HEK293 cells ( Figure 3 ) and ARPE-19 cells ( Figure 4 ). Therefore, using BCD1 as a control, transgenic constructs BCD8, BCD9, BCD15, BCD16, and BCD17 were selected for further evaluation. Western blot (WB) results were Figure 5 and Figure 6 The grayscale values ​​of all bands are calculated, analyzed and normalized to BCD1 ( Figure 5 and Figure 6 , small picture below).

[0071] Based on Western blot results, it was determined that BCD8 exhibited the highest CYP4V2 protein expression level in HEK293 cells ( Figure 5 ), and BCD16 showed the highest CYP4V2 protein expression level in ARPE-19 cells ( Figure 6 ).

[0072] Subsequently, BCD1, BCD8, and BCD16 were packaged into replication-deficient AAV8. AAV8-BCD1, AAV8-BCD8, and AAV8-BCD16 virus particles were used to generate a single-cell hybridoma at an MOI of 5 × 10 in the presence of 2 mM hydroxyurea. 5 Transduction of ARPE-19.

[0073] AAV transduction experiments showed that AAV8-BCD16 had the highest expression level ( Figure 7 ).

[0074] References

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Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO: 16 and encoding a human CYP4V2 polypeptide. 2 . The isolated nucleic acid molecule according to claim 1 , further comprising a promoter operably linked to the 5′ of the nucleotide sequence encoding CYP4V2.

3. The isolated nucleic acid molecule of claim 2, wherein the promoter is a CAG promoter.

4. The isolated nucleic acid molecule of claim 1, further comprising a polyadenylation sequence 3' to the nucleotide sequence encoding CYP4V2.

5. The isolated nucleic acid molecule of claim 4, wherein the polyadenylation sequence is bovine growth hormone poly A, synthetic poly A, or simian virus 40 poly A.

6. The isolated nucleic acid molecule of claim 5, wherein the polyadenylation sequence is simian virus 40 poly A.

7. The isolated nucleic acid molecule of claim 1, further comprising a Woodchuck Hepatitis Virus post-transcriptional regulatory element.

8. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence shown in SEQ ID NO:

33.

9. A recombinant AAV vector comprising the nucleic acid molecule according to any one of claims 1 to 8.

10. The recombinant AAV vector of claim 9, comprising two ITRs.

11. The recombinant AAV vector of claim 10, comprising two ITRs derived from AAV2.

12. A viral particle comprising the recombinant AAV vector according to claim 9 packaged into an AAV capsid.

13. The viral particle of claim 12, wherein the AAV capsid is an AAV8 capsid.

14. A pharmaceutical composition comprising the viral particle according to claim 12 or claim 13 and a pharmaceutically acceptable excipient.

15. Use of the rAAV vector according to claim 9 in the manufacture of a medicament for treating crystalline retinal degeneration.

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