Adenine base editor for DFNB9 hearing loss repair

Abstract

The invention discloses an adenine base editor for DFNB9 hearing loss repair, and belongs to the field of gene therapy of hearing repair. The fusion protein comprises nNme2Cas9, and adenine deaminase TadA-8e or TadA-8e-N108Q (adenine deaminase) or TadA-8e-N108Q-L145T (adenine deaminase). The invention further discloses a preparation method of the adenine deaminase. The amino acid sequences of the TadA-8e, the TadA-8e-N108Q and the TadA-8e-N108Q-L145T are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 3. The fusion protein and a guide RNA form a base editor. Compared with the prior art, the base editor is used as a treatment means, severe deafness can be recovered to a normal level, and the effect can be maintained for at least six months after treatment. In addition, the treatment window period is relatively long, and the method has clinical transformation significance.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy for hearing restoration, and specifically to an adenine base editor for repairing DFNB9 hearing loss. Background Art

[0002] Hearing loss is one of the most common sensory disorders, affecting approximately 466 million people worldwide (approximately 5% of the global population), including 34 million children. Among them, the OTOF gene encodes the OTOF protein, which is associated with profound recessive hearing loss associated with the auditory neuropathy (AN) type DFNB9. Currently, more than 100 OTOF mutations are known to cause hearing loss. Currently, cochlear implants (CI) are the main treatment for DFNB9 patients. However, CI requires lifelong management and regular surgical interventions for maintenance. Recent advances in gene editing have opened up promising new avenues for treating genetic diseases such as DFNB9.

[0003] In previous studies, the inventors successfully demonstrated the feasibility of gene replacement therapy in a clinical setting, restoring hearing in DFNB9 patients. Some of these patients achieved near-normal hearing thresholds after treatment. However, gene replacement therapy does not directly correct the underlying mutation in the OTOF gene, and its therapeutic effects may weaken over time. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention proposes an adenine base editor for repairing DFNB9 hearing loss.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to a fusion protein comprising:

[0007] nNme2Cas9, and

[0008] Adenine deaminase TadA-8e or TadA-8e-N108Q or TadA-8e-N108Q-L145T, the amino acid sequences of TadA-8e, TadA-8e-N108Q, TadA-8e-N108Q-L145T are: SEQ ID NO.1~3 respectively.

[0009] Optionally, the fusion protein is subjected to split intein-mediated protein trans-splicing, comprising the following steps: co-transfecting cells with N-terminal and C-terminal plasmids overexpressing Nme2Cas9, wherein the full length of Nme2Cas9 is SEQ ID NO.4, and the cleavage site 2 of protein trans-splicing is located between positions 510 and 511 of SEQ ID NO.4.

[0010] Optionally, the amino acid sequence of the intein is SEQ ID NO.5 and SEQ ID NO.6.

[0011] The second aspect of the present invention relates to a base editor, comprising the above-mentioned fusion protein and a guide RNA.

[0012] Optionally, the nucleic acid sequence of the guide RNA is: SEQ ID NO.7.

[0013] The third aspect of the present invention relates to an expression vector comprising:

[0014] A nucleic acid encoding the above-mentioned fusion protein; and a nucleic acid encoding the above-mentioned guide RNA.

[0015] Optionally, the expression vector is AAV.

[0016] The fourth aspect of the present invention relates to a drug for treating hearing loss, comprising: the above-mentioned fusion protein, base editor or expression vector.

[0017] Optionally, the drug is an injection.

[0018] The fifth aspect of the present invention relates to the use of the above-mentioned fusion protein, base editor or expression vector in the preparation of a drug for treating hearing loss.

[0019] Beneficial effects of the present invention:

[0020] The present invention utilizes split intein-mediated protein trans-splicing to inject Nme2ABE8e into adult Otof through the semicircular canal. 2815C>T / 2815C>T After the mouse cochlea was implanted, the hearing of the treated mice recovered from severe deafness to normal levels, and the effect was maintained for at least six months after treatment. In addition, the treatment window period is longer, which is more meaningful for clinical transformation. In short, the present invention provides a safe, effective and stable treatment method for DFNB9, and may be extended to treat other hereditary hearing losses. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the accompanying drawings.

[0022] Figure 1 .Nme2ABE8e vs Otof 2815C>T / 2815C>TEditing test of point mutations. (A) Schematic diagram of ABE: purple is Cas9 nickase (nSpCas9-NG, nNme2Cas9), green is TadA adenine deaminase (TadA-8e, TadA-8e-N108Q, TadA-8e-N108Q-L145T), orange is sgRNA, and blue is PAM. (B) Optimized Nme2ABE tool: Adding DNA double-strand binding protein HMG-D to Nme2ABE8e can construct eeNme2ABE8e, and adding DNA single-strand binding protein Rad51 to Nme2ABE8e can construct hyNme2ABE8e. Nme2ABE8e-N108Q is replaced by TadA8e-N108Q in Nme2ABE8e. Nme2ABE9 is replaced by TadA-8e-N108Q-L145T in Nme2ABE8e. (C) Editing efficiency of the Nme2ABE toolbox at target sites.

[0023] Figure 2 .Nme2ABE8e therapeutic strategy selection and testing. (A) The three-dimensional structure of the Nme2Cas9 protein predicted using AlphaFold, with the location of the cleavage site in the linker region highlighted (red sphere). The different protein domains are color-coded. (BC) Western blot analysis shows intein-mediated recombinant expression of nine split Nme2ABE8e variants. (D) Heat map showing the editing efficiency of variant 2 of split Nme2ABE8e at sgRNA1.

[0024] Figure 3 Otof 2815C>T / 2815C>T Hearing recovery of mice. (A) Average Click ABR thresholds measured at 1 week, 1 month, and 6 months after injection in P30 mice. (B) Average Tone-burst ABR thresholds measured at the same time points (1 week, N=10; 1 month, N=8; 6 months, N=3).

[0025] (C) ABR wave I latency (upper) and amplitude (lower) at 4, 8, 12, 16, 24, and 32 kHz in WT mice (black), contralateral ears (blue), and AAV-injected ears (red) (N=6) at 1 month after surgery. (D) ABR thresholds in P90-injected mice 1 week after injection (injected ears, N=3). (E) ABR thresholds in P90-injected mice 1 month after injection (injected ears, N=3). Error bars represent standard error of the mean (SEM). Statistical significance was determined by Student's t-test: *p<0.05, **p<0.01, ****p<0.0001, and "ns" indicate no significant difference. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the examples of this application, the antisense strand of the Otof gene was focused on, and two Cas proteins, nNme2Cas9 and nSpCas9-NG, were identified at the mutation site. In order to improve editing accuracy, the standard TadA-8e enzyme was replaced by improved adenine deaminase TadA-8e-N108Q and TadA-8e-N108Q-L145T, resulting in Nme2ABE8e-N108Q and Nme2ABE9. These modifications are intended to optimize editing accuracy and activity.

[0028] The amino acid sequences involved above specifically include:

[0029] TadA-8e (SEQ ID NO.1):

[0030] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0031] TadA-8e-N108Q (SEQ ID NO. 2):

[0032] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRQSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0033] TadA-8e-N108Q-L145T(SEQ ID NO.3):

[0034] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRQSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALTCDFYRMPRQVFNAQKKAQSSIN

[0035] To evaluate the efficiency of these base editors, Otof 2815C>T The mutation was integrated into the genome of HEK293T cells, and stable cell lines were selected using red fluorescent labeling and puromycin selection. After these cells were co-transfected with ABE-sgRNA complexes (a combination of fusion protein and sgRNA), the cells were sorted by flow cytometry and subjected to next-generation sequencing (NGS) to evaluate the editing efficiency. The results showed that Nme2ABE8e had the highest editing efficiency on sgRNA1, with a success rate of 68.8%.

[0036] The expression of full-length Nme2Cas9 in cochlear hair cells was achieved by split intein-mediated protein trans-splicing. The selection of the split site was based on the requirement that the N-terminal amino acid should be Cys, Ser or Thr. These sites were further refined using Alphafold protein structure prediction to ensure that all split sites were located in the junction region. This process determined nine optimal split site pairs. Next, cells (untreated blank cells) were co-transfected with two plasmids encoding split Nme2Cas9 variants. After 48 hours, protein extraction and Western blotting were performed to assess expression levels. The results showed that the protein level produced by cleavage site 2 (between positions 510 and 511 of SEQ IDNO.4) was comparable to that of full-length Nme2Cas9. The cleavage plasmid was then co-transfected into the stable cell line constructed above, and after three days of sorting, genomic DNA extraction, PCR amplification and deep sequencing were performed to assess editing efficiency. It was found that the editing efficiency of cleavage site 2 was indeed high, at 67.8%, which was comparable to that of the complete Nme2Cas9. Based on Western blotting and editing efficiency results, cleavage Nme2ABE8e variant 2 was selected for further therapeutic development.

[0037] Specifically, the object of splitting or cleavage described in this embodiment, Nme2Cas9 (SEQ ID NO.4), has a sequence of:

[0038]

[0039] Other cleavage sites include:

[0040] Cleavage site 1: between positions 495 and 496 of SEQ ID NO.4;

[0041] Cleavage site 3: between positions 548 and 549 of SEQ ID NO.4;

[0042] Cleavage site 4: between positions 564 and 565 of SEQ ID NO.4;

[0043] Cleavage site 5: between positions 567 and 568 of SEQ ID NO.4;

[0044] Cleavage site 6: between positions 591 and 592 of SEQ ID NO.4;

[0045] Cleavage site 7: between positions 597 and 598 of SEQ ID NO.4;

[0046] Cleavage site 8: between positions 616 and 617 of SEQ ID NO.4;

[0047] Cleavage site 9: between positions 640 and 641 of SEQ ID NO.4.

[0048] The sequence of the intein described in this example is:

[0049] Rm-intein-N (SEQ ID NO.5):

[0050] NCLAGDTLITLADGRRVPIRELVSQQNFSVWALNPQTYRLERARVSRAFCTGIKPVYRLLTTRLGRSIRATANHRFLTPQGWKRVDELQPGDYLALPRRIPTAS

[0051] Rm-intein-C (SEQ ID NO.6):

[0052] AAACPELRQLAQSDVYWDPIVSIEPDGVEEVFDLTVPGPHNFVANDIIAHN

[0053] The sequence of the sgRNA described in this example (SEQ ID NO.7) is:

[0054] GAGGAGGTCAAGGCAGCCTAAGGC.

[0055] To evaluate the effect of Nme2ABE8e on adult Otof2815C>T / 2815C>T To investigate the effect of AAV on hearing recovery in mice, a 1:1 dual AAV mixture (corresponding to the two ends of Nme2Cas9-N + Nme2Cas9-C removed from the cleavage site 2) was injected into the cochlea of ​​P30 homozygous mutant mice through the posterior semicircular canal. Click-ABR and Tone-burst ABR were then used to monitor hearing recovery at intervals of 1 week, 1 month, and 6 months after surgery. The control group included the contralateral ear and WT mice. Click ABR results showed that Otof 2815C>T / 2815C>T The hearing of the mice returned to normal levels within 1 week after surgery and remained stable for at least six months. The short tone frequency ABR results showed that the ABR thresholds of the AAV-injected ear increased by 35dB at 4kHz, 45dB at 8kHz, 50dB at 12kHz, 40dB at 16kHz, 35dB at 24kHz, and about 30dB at 32kHz within 1 month after surgery compared with the contralateral ear (thresholds of 85-90dB for all frequencies). These improvements were not significantly different from those observed in WT C57BL / 6J mice with the same genetic background. At a stimulation intensity of 90dB, the ABR wave I amplitude of the treated ear returned to WT levels at 4, 8, and 12kHz, and the wave I latency also returned to normal levels. Long-term ABR (6 months after surgery) showed that the hearing recovery of the AAV-injected ear was stable at all frequencies (4K-32KHz). These findings suggest that precision gene therapy can effectively restore Otof. 2815C>T / 2815C>T The hearing of the mice returned to normal levels, and this recovery continued over time. Subsequently, to evaluate the hearing recovery after injection of Nme2ABE8e in older point mutant mice, the editing tool was injected at P90, and ABR tests were performed 1 week and 1 month after injection to monitor the recovery. At both time points, Click ABR and Tone-burst ABR showed significant improvement compared to the contralateral ear.

[0056] In summary, this application screened out an adenine to guanine base editor by combining Nme2-Cas9 with ABE8e. This editor effectively corrected the pathogenic Otof mutation c.2815C>T (p.Gln939*) in adult mice, with better results than other base editors. Using cleavage intein-mediated protein trans-splicing, Nme2ABE8e was delivered to adult Otof cells by retrosemicircular canal injection. 2815C>T / 2815C>TThe cochlea of ​​mice. The treated mice recovered hearing from severe deafness to normal hearing levels, and sustained effects were observed for at least six months after treatment. In addition, the treatment window was long, which is more meaningful for clinical translation. These findings highlight the safety and effectiveness of this base editing strategy as a treatment option for DFNB9 and lay the foundation for its potential application in treating other genetic hearing losses.

[0057] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0058] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A fusion protein, characterized in that include: nNme2Cas9, and Adenine deaminase TadA-8e or TadA-8e-N108Q or TadA-8e-N108Q-L145T, the amino acid sequences of TadA-8e, TadA-8e-N108Q, TadA-8e-N108Q-L145T are SEQ ID NOs. 1 to 3, respectively.

2. The fusion protein according to claim 1, characterized in that The fusion protein undergoes split intein-mediated protein trans-splicing. The method comprises the following steps: co-transfecting cells with N-terminal and C-terminal plasmids over-expressing Nme2Cas9, wherein the full length of the Nme2Cas9 is SEQ ID NO.4, and the cleavage site of protein trans-splicing is between positions 510 and 511 of SEQ ID NO.

4.

3. The fusion protein according to claim 2, characterized in that The amino acid sequences of the intein are SEQ ID NO.5 and SEQ ID NO.

6.

4. A base editor, comprising the fusion protein according to any one of claims 1 to 3, and a guide RNA.

5. The base editor according to claim 4, characterized in that The nucleic acid sequence of the guide RNA is: SEQ ID NO.

7.

6. An expression vector, characterized in that: include: A nucleic acid encoding the fusion protein according to any one of claims 1 to 3; and a nucleic acid encoding the guide RNA according to claim 4 or 5.

7. The expression vector according to claim 6, characterized in that The expression vector is AAV.

8. A drug for treating hearing loss, characterized in that: include: The fusion protein of any one of claims 1 to 3, the base editor of claim 4 or 5, or the expression vector of claim 6 or 7.

9. The drug for treating hearing loss according to claim 8, characterized in that: The medicine is an injection.

10. Use of the fusion protein of any one of claims 1 to 3, the base editor of claim 4 or 5, or the expression vector of claim 6 or 7 in the preparation of a drug for treating hearing loss.

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