WO2023129095A1

WO2023129095A1 - Crispr-pe system for retinol dehydrogenase 12 (rdh12) gene mutations for use in the treatment of retinitis pigmentosa (rp) disease

Info

Publication number: WO2023129095A1
Application number: PCT/TR2022/051691
Authority: WO
Inventors: Gorkem AKGUL; Ali Dostcan AKYAR; Batuhan YOLVER; Cihan TASTAN
Original assignee: T.C. Uskudar Universitesi
Priority date: 2021-12-31
Filing date: 2022-12-29
Publication date: 2023-07-06
Also published as: TR2021022284A2

Abstract

The invention relates to prime editing guide RNA (pegRNA) sequences, CRISPR-PE (Regularly Interrupted Palindromic Repeat Sets-Prime Editing) system and the method of transferring the aforementioned pegRNA sequences to the target region with neural lentivirus by integrating them into the CRISPR-PE system and thereby correcting the mutations for correction of pathogenic mutations, particularly the C146T/A and 778delG mutations in Retinitis Pigmentosa disease on the retinol dehydrogenase 12(RDH12) gene, for use in the treatment of Retinitis Pigmentosa (RP) disease. With the invention, PegRNA sequences that has a low indel mutation risk, high reproductive rate, and genome integration capability, and provides correction of pathogenic mutations on the RDH12 gene in Retinitis Pigmentosa, especially the C146T/A and 778delG mutations on the RDH12 gene and the CRISPR-PE system containing these sequences and the lentiviral vector for use in the treatment of Retinitis Pigmentosa with the invention are provided.

Description

CRISPR-PE SYSTEM FOR RETINOL DEHYDROGENASE 12 (RDH12) GENE MUTATIONS FOR USE IN THE TREATMENT OF RETINITIS PIGMENTOSA (RP) DISEASE

Technical Field of the Invention

The invention relates to prime editing guide RNA (pegRNA) sequences, CRISPR- PE (Regularly Interrupted Palindromic Repeat Sets-Prime Editing) system and the method of transferring the aforementioned pegRNA sequences to the target region with neural lentivirus by integrating them into the CRISPR-PE system and thereby correcting the mutations for correction of pathogenic mutations, particularly the C146T/A and 778delG mutations in Retinitis Pigmentosa disease on the retinol dehydrogenase '\2(RDH12) gene, for use in the treatment of Retinitis Pigmentosa (RP) disease.

State of the Art

Retinitis Pigmentosa (RP) is a chronic inherited eye disease characterized by rod and cone deficiency and gradual degeneration of light-sensitive retinal cells covering the back of the eye. RP can result from both homozygous and heterozygous mutations. For example, RP can exist in various forms such as autosomal dominant RP (adRP), autosomal recessive RP (arRP), or X-linked RP (X-LRP). Although treatment options for RP are limited, there is no approved treatment available in the state of the art that can stop or reverse the progression of RP.

Retinitis Pigmentosa is known to be associated with the retinol dehydrogenase 12 (RDH12) gene. RDH12 is a member of the short chain dehydrogenase/reductase enzyme family. Diseases associated with RDH12 gene include Leber Congenital Amaurosis 13 and Retinitis Pigmentosa. In the retina, RDH12 plays a critical role in reducing toxic retinaldehydes produced by visual loop activity, which is essential for the light response of photoreceptor cells. Individuals with RDH12 deficiency suffer from retinal degeneration affecting both rods and cones. It is known that RP disease occurs due to many pathogenic mutations on the RDH12 gene, the C146T/A and 778delG mutations being in the first place.

Today, one of the technologies used for the development of RP therapy is CRISPR. Like many genetic diseases and unwanted traits, RP is caused by base pair changes in genomic DNA. Base editing (BE), the newest application of CRISPR-Cas-based technologies, can generate point mutations directly in cell DNA without causing a double-stranded DNA break (DSB). Two classes of DNA base modifiers have been identified so far, cytosine base modifiers (CBEs) and adenine base modifiers (ABEs). Recently, a new CRISPR-based strategy, prime editing (PE) system, has been developed for precision genome editing, which enables the transfer of various genomic changes directly to target sites without requiring double-strand breaks (DSBs) or donor templates. Prime editing (PE) has extended the CRISPR-based-editing system with many types of mutations, and this approach includes two key components. The first is a catalytically degraded Cas9 nicase fused to the reverse transcriptase, and the second is a multifunctional prime editing guide RNA (pegRNA) that specifies the target site and also serves as a template for reverse transcription (RT). pegRNAs are similar to standard single guide RNAs (sgRNAs), but have an additional customizable extension at the 3' end. The 3' end consists of an RT template encoding the desired rearrangement and a primer binding site (PBS) that annealing to the target genomic region to initiate the RT reaction 2.

In the prior art, treatment studies for RDH12 gene mutations associated with Retinitis Pigmentosa and Leber Congenital Amarosis have been limited. The gene editing method generally used in studies is CRISPR-CAS9 and includes the transfer of genes with AAV2/8 heterovectors. Both the high off-target (undesired interference outside the targeted region on the genome) score of CRISPR-CAS9 and the passive nature of AAV vectors in genome integration cause frequent treatment expenditures. In addition, CRISPR-CAS9 and AAV2/8 heterovectors reduce the effectiveness of current treatment trials. It is known that the CRISPR-PE system reduces the indel mutation risk 270 times compared to conventional CAS9-H DR-based treatment strategies [1], In addition, unlike CRISPR-BE technology, which corrects 4 different transition (point) mutations without DNA strand breakage, the CRISPR-PE strategy precisely regulates 12 different transition (point) mutations [2] and thus CRISPR-PE provides a more effective outlook than other gene editing strategies. The viral used to deliver CRISPR systems is generally AAV-based. This causes the treatment strategy not to be long-lasting and shortens the duration of action. Lentiviruses (LV), another option used in the delivery of CRISPR systems, increase the continuity of treatment in terms of their genome integration ability and high reproductive rate. LV vectors are retroviral vectors that can infect nondividing cells and typically produce high viral titres. Therefore, lentiviruses are considered as one of the most effective agents used in gene transfer. AAVs, on the other hand, have a major disadvantage that limits the size of the expression cassette to a maximum of 4.5 kb, while a lentiviral vector can carry a 10 kb insert.

Prior art CA3130515A1 patent application describes compositions and methods for CRISPR/RNA based nuclease for the treatment of RHO-associated retinitis pigmentosa, for example, autosomal dominant retinitis pigmentosa (adRP). Here, the guide RNA (gRNA) molecule that binds to the target sequence of the RHO gene is mentioned. Another prior art document, WO2019102381 A1 , describes materials and methods for the treatment of autosomal dominant Retinitis Pigmentosa (RP) disease both ex vivo and in vivo. However, the documents in the prior art are insufficient for use in the treatment of Retinitis Pigmentosa in terms of the gene regions they target, and the systems used.

Due to the facts that CRISPR systems in the prior art developed for use in the treatment of Retinitis Pigmentosa are insufficient to correct pathogenic mutations, increase the risk of indel mutations, have a high risk of causing unwanted mutations outside the targeted region on the genome, involve AAV delivery in gene delivery and are passive in genome integration of AAV vectors, there is a need to develop CRISPR systems which can correct pathogenic mutations specific to Retinitis Pigmentosa, have low indel mutation risk, high reproductive rate and genome integration, for use in the treatment of Retinitis Pigmentosa,

Brief Description of the Invention

In the invention, prime editing guide RNA (pegRNA) sequences for correction of pathogenic mutations on the retinol dehydrogenase 12 (RDH12) gene, CRISPR- PE (Regularly Interleaved Palindromic Repeat Sets-Primary Arrangement) system and the method of transferring the aforementioned pegRNA sequences to the target region with neural lentivirus by integrating them into the CRISPR-PE system and thereby correcting the mutations are explained.

The first object of the invention is to provide pegRNA sequences for the correction of pathogenic mutations in the RDH12 gene for use in the treatment of Retinitis Pigmentosa. In the invention, pegRNA sequences are designed for the correction of pathogenic mutations in Retinitis Pigmentosaon the RDH12gene, the C146T/A and 778delG mutations being in the first place. Then, these pegRNA sequences are integrated into the CRISPR-PE system and transferred to the target region with neural lentivirus, and the mutations are corrected. Mutant RDH12 specific pegRNAs enable the CRISPR enzyme to recognize the region to cut, and photoreceptor and neuron-specific promoter sequences are the nucleotide sequences in which transcription (mRNA production) begins, and enable the CRISPR system to initiate transcription in the targeted gene. Each pegRNA disclosed within the scope of the invention is specific for the pathogenic mutations of its corresponding RDH12 gene and is used in the personalized genetic treatment of retinitis pigmentosa patients who have one or more of the mutations at the relevant position.

Another aim of the invention is to provide CRISPR systems with low indel mutation risk, high reproductive rate and genome integration capability to be used in the treatment of Retinitis Pigmentosa. With the neural lentivirus with a high reproductive rate used in the invention, the CRISPR-PE coding system is transferred to the relevant gene region for the correction of pathogenic mutations in the RDH 12 target region. Photoreceptor-specific neural lentiviruses used within the scope of the invention provide the transport of the CRISPR-PE system to the photoreceptor cells, the integration of the photoreceptor cells into the genome and the initiation of the genetic editing process.

With the invention, PegRNA sequences that has a low indel mutation risk, high reproductive rate and genome integration capability, and provides correction of pathogenic mutations on the RDH12 gene in Retinitis Pigmentosa, especially the C146T/A and 778delG mutations on the RDH12 gene and the CRISPR-PE system containing these sequences and the lentiviral vector for use in the treatment of Retinitis Pigmentosa with the invention are provided.

Detailed Description of the Invention

The invention relates to prime editing guide RNA (pegRNA) sequences, CRISPR- PE (Regularly Interrupted Palindromic Repeat Sets-Prime Editing) system and the method of transferring the aforementioned pegRNA sequences to the target region with neural lentivirus by integrating them into the CRISPR-PE system and thereby correcting the mutations for correction of pathogenic mutations, particularly the C146T/A and 778delG mutations in Retinitis Pigmentosa disease on the retinol dehydrogenase '\2(RDH12) gene, for use in the treatment of Retinitis Pigmentosa disease.

In order to obtain the CRISPR-PE system, which is the subject of the invention, first of all, pegRNA designs were made to edit/repair the pathogenic mutations in the RDH12 gene, then the RDH12 specific CRISPR-PE components were brought together to obtain the vector. Afterwards, neural lentiviruses encoding the RDH12 specific CRISPR-PE system were produced and transduced with ex- vivo and in-vivo CRISPR-PE positive neural lentiviruses.

First, pegIT software was used to design pegRNAs to correct/repair pathogenic mutations in the RDH12 gene and 51 pegRNAs were designed. Said pegRNA sequences are presented in the sequence list, and these pegRNAs play an important role in guiding the CRISPR enzyme and are mutation specific. These selected mutations are pathogenic mutations and were obtained from the ClinVar (NCBI) database. These pathogenic mutations are: at least one mutation chosen between C146T, C146A, 778delG, C63_66del, T125C, C138T, G148A, G149A, T152A, C164T, C184T, A195C, G226C, G226A, G226T, C295A, C300T, C316T, C377T, G379T, T393A, T437A, T446C, G448+1A, 488delC, 496delC, T523C, C524A, C565T, C570T, 580dup, C582G, A599G, A599C, C609A, G658+1A, T659-12C, A659-2T, C688G, 759dup, 759delC, 784dup, 806_810del, 812_813del, T821 C, T848+2C, 869dup, T869G, C883T and *54GC mutations.

Each pegRNA produced is specific for the pathogenic mutations (ClinVar) of the corresponding RDH12 gene in Table 1 and is designed for use in personalised genetic therapy of retinitis pigmentosa patients with one or more of these mutations. Multiple mutations occurring at the same position with a pegRNA can be repaired and there is only one pegRNA in a vector. Accession number for wild type (wt) RDH 12 mentioned in the invention: KR710645 (GenBank), transcription number: NM_152443.3.

The prime editing (PE) system used in the invention is a technology that can enable genomic edits without double-stranded DNA breaks (DSBs) or donor DNA. pegRNAs simultaneously encode both guide and regulatory template sequences. 51 pegRNAs within the scope of the invention consist of a sequence of pegRNA complementary to target sites that directs nCas9 to its target sequence, a reverse transcriptase (RT) sequence encoding desired sequence changes/edits, a primer binding sequence (PBS) annealing to the target genomic region to initiate RT reaction 2 and an sgRNA scaffold sequence required for Cas- binding. The mutant RDH12 specific pegRNAs in the invention enable the CRISPR enzyme to recognize the region to be cut and to make the desired mutation correction/repair.

The designed pegRNA sequences are specific for the mutation to which they are targeted. The types of mutations targeted by pegRNAs are divided into types such as duplication, deletion and base change mutation, as indicated in the mutation names. Technically, it is based on the principle of redeploying the base that was deleted in the deletion to the correct location while one of the nucleotides paired in duplication is deleted. In base change modification, on the other hand, the principle of replacing the wrong base with the correct base works. Bases indicated in small letters in the pegRNA table indicate the target region of that pegRNA. The numbers in the mutation nomenclature corresponding to the sequences in the pegRNA table indicate the location where that mutation occurred. For example, in the 778delG mutation, the G (Guanine) base appears to be deleted at the 778th nucleotide. With the pegRNA designed specifically for the 778delG mutation, the deleted G base is re-inserted at position 778 using the CRISPR-PE system. pegRNA sequences designed for cDNA.146. T— >C, cDNA.146. A^C, and cDNA.778. G base addition modifications (SEQ ID NO:48-51 ) and other mutations are presented in the (SEQ ID NO: 1 -47) sequence list and in Table 1.

Table 1. pegRNA sequences designed for cDNA.146. T— >C, cDNA.146. A— >C and cDNA.778. G base insertion modifications and other mutations (Sequence number/SEQ ID NO:1 -51 ).

After mutant RDH12 specific pegRNAs are designed, a vector is created so that these pegRNAs can be transferred to target sites. RDH12-specific CRISPR-PE components, the prime editing (PE) enzyme designed for the treatment of RP, pegRNAs and promoter sequences required for initiation of transcription in the gene are brought together and a vector containing these components is obtained.

The PE enzyme, which is a reverse transcriptase enzyme fused to Cas9 endonuclease, which functions as a catalytic nicase in the vector content, is responsible for cutting the DNA chain. The PE enzyme used within the scope of the invention is encoded by one of the CMV, RHO, hGRK, hRP1 , photoreceptor and neural lentivirus specific promoters. For this, a promoter selected from CMV, RHO, hGRK and hRP1 is combined with the PE enzyme. These promoters make transcription more effective. The PE enzyme can be encoded by all of the aforementioned promoters (CMV, RHO, hGRK and hRP1 ). The PE enzyme is combined with each promoter separately.

The vector also contains the pU6 promoter, to which RNA polymerase III binds, which is responsible for the transcription of pegRNAs. Neural lentivirus vectors were used as vectors and all mentioned components were cloned into the same neural lentiviral vector. Because lentiviruses have the ability to transfer significant amounts of genetic information to the host cell, the use of these vectors is considered one of the most effective methods of gene delivery in gene therapy. The lentiviral genome consists mainly of the gag/pol and env genes. All structural proteins are encoded by the gag domain, such as the membrane-associated matrix protein, the core-forming capsid protein, and the nucleocapsid protein that binds to viral RNA. The pol gene provides the formation of viral enzymes such as protease, reversible transcriptase, and integrase. The env gene encodes the envelope.

Within the neural lentiviral vector/plasmid, from 5'-LTR (long terminal repeats) to 3'LTR there respectively are

• photoreceptor and neuron specific promoter,

• CRISPR prime editing (PE) enzyme coupled with a promoter selected from the CMV, RHO, hGRK and hRP1 promoters,

• pU6 promoter,

• Mutant RDH12 specific pegRNA

. The gag, pol and env parts, which are outside the 5'-LTR-Photoreceptor specific promoter- CRISPR PE Enzyme- Pu6 Promoter- pegRNA-3'LTR vector sequence in the vector, are in the lentiviral vector as known in the prior art and are used in the synthesis step. In the production of neural lentiviruses encoding the RDH12 specific CRISPR-PE system, pHIV1 encoding HIV1 viruses or pEV53D encoding EIAV viruses are used viral plasmids. The pEV53D plasmid was chosen because it encodes pH IV and EIAV viruses. When producing these plasmids encoding CRISPR-PE, plasmid DNAs encoding envelope proteins (FLIG-B2 or FLIG-E) that bind specifically to neuron cells are used. FLIG-B2 and FLIG-E are capsid proteins used to make virus attachment more effective. The invention uses HIV or EIAV plasmid pseudo typed separately for FLIG-B2 and FLIG-E. However, other lentiviruses encoding FLIG-B2 and FLIG-E can also be used to carry out the invention.

Neural lentiviruses act as biological agents and integrate the CRISPR-PE system into the genome of photoreceptor cells, thus enabling the genetic editing process to begin.

The CRISPR-PE system packaged into neural lentiviruses is used for the modification of target photoreceptor cells ex vivo and in vivo. After transmission, the viral agent enters the photoreceptor cells and encodes the CRISPR-PE system of the invention. Then, using the pegRNA sequences in the CRISPR-PE system, the target mutations in the RDH12 gene are repaired with the PE enzyme expressed only by the promoters that initiate transcription in neural cells. Following mutation repair, the functional RDH12 protein will be optimally expressed.

References

1. Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149-157. https://doi.org/10.1038/s41586-019- 1711-4

2. Kantor, A., McClements, M. E., & MacLaren, R. E. (2020). CRISPR-Cas9 DNA Base-Editing and Prime-Editing. International journal of molecular sciences, 21 (17), 6240. https://doi.org/10.3390/ijms21176240

Claims

CLAIMS Prime editing guide RNA (pegRNA) for use in the Regularly Split Palindromic Repeat Sets-Prime Editing (CRISPR-PE) system for correction of pathogenic mutations on the retinol dehydrogenase 12 (RDH12) gene in Retinitis Pigmentosa disease, comprising a nucleotide sequence selected from the sequences of SEQ ID NO: 1 -51. pegRNA according to claim 1 , wherein said pathogenic mutations in the retinol dehydrogenase 12 (RDH12) gene in Retinitis Pigmentosa disease are at least one mutation selected from C146T, C146A, 778delG, C63_66del, T125C, C138T, G148A, G149A, T152A, C164T, C184T, A195C, G226C, G226A, G226T, C295A, C300T, C316T, C377T, G379T, T393A, T437A, T446C, G448+1A, 488delC, 496delC, T523C, C524A, C565T, C570T, 580dup, C582G, A599G, A599C, C609A, G658+1A, T659-12C, A659-2T, C688G, 759dup, 759delC, 784dup, 806_810del, 812_813del, T821 C, T848+2C, 869dup, T869G, C883T and *54GC mutations as duplication (dup), deletion (del) or base change mutations. A lentiviral vector for use in correcting pathogenic mutations on the RDH12 gene in Retinitis Pigmentosa disease, comprising, from 5'-LTR (long terminal repeats) to - 3'LTR, respectively, i. photoreceptor and neuron specific promoter, ii. CRISPR prime editing (PE) enzyme coupled with the CMV, RHO, hGRK and hRPI promoters, iii. pU6 promoter, iv. Mutant RDH12 specific pegRNA having a nucleotide sequence selected from SEQ ID NOs:1-51. A lentiviral vector according to claim 3, wherein said pathogenic mutations in the retinol dehydrogenase 12 (RDH12) gene in Retinitis Pigmentosa disease are at least one mutation selected from C146T, C146A, 778delG, C63_66del, T125C, C138T, G148A, G149A, T152A, C164T, C184T, A195C, G226C, G226A, G226T, C295A, C300T, C316T, C377T, G379T, T393A, T437A, T446C, G448+1A, 488delC, 496delC, T523C, C524A, C565T, C570T, 580dup, C582G, A599G, A599C, C609A, G658+1A, T659-12C, A659-2T, C688G, 759dup, 759delC, 784dup, 806_810del, 812_813del, T821 C, T848+2C, 869dup, T869G, C883T and *54GC mutations as duplication (dup), deletion (del) or base change mutations.

5. A lentiviral plasmid for use in correcting pathogenic mutations on the RDH12 gene in Retinitis Pigmentosa disease, comprising, from 5'-LTR (long terminal repeats) to - 3'LTR, respectively, v. photoreceptor and neuron specific promoter, vi. CRISPR prime editing (PE) enzyme coupled with the CMV, RHO, hGRK and hRPI promoters, vii. pU6 promoter, viii. Mutant RDH12 specific pegRNAhaving a nucleotide sequence selected from SEQ ID NOs:1-51.

6. A lentiviral plasmid according to Claim 5, wherein said pathogenic mutations in the retinol dehydrogenase 12 (RDH12) gene in Retinitis Pigmentosa disease are at least one mutation selected from C146T, C146A, 778delG, C63_66del, T125C, C138T, G148A, G149A, T152A, C164T, C184T, A195C, G226C, G226A, G226T, C295A, C300T, C316T, C377T, G379T, T393A, T437A, T446C, G448+1A, 488delC, 496delC, T523C, C524A, C565T, C570T, 580dup, C582G, A599G, A599C, C609A, G658+1A, T659-12C, A659-2T, C688G, 759dup, 759delC, 784dup, 806_810del, 812_813del, T821 C, T848+2C, 869dup, T869G, C883T and *54GC mutations as duplication (dup), deletion (del) or base change mutations.

7. A lentiviral plasmid according to Claim 5, wherein said lentiviral plasmid is pHIV1 plasmid encoding HIV1 viruses or pEV53D plasmid encoding EIAV viruses.

8. A lentiviral plasmid according to Claim 7, wherein said pHIV1 or pEV53D plasmid is a plasmid encoding FLIG-B2 or FLIG-E.