CN117866970A

CN117866970A - Genetically engineered constructs for choroidal disorders

Info

Publication number: CN117866970A
Application number: CN202311288474.6A
Authority: CN
Inventors: 沈吟
Original assignee: Zhongmou Medical Technology Wuhan Co ltd
Current assignee: Zhongmou Medical Technology Wuhan Co ltd
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-04-12

Abstract

Engineered nucleic acid molecules are provided for use in the treatment of choroidal disorders, which can express a CHM protein or fragment thereof. And, further provided are recombinant AAV comprising the engineered nucleic acid molecules and uses thereof.

Description

Genetically engineered constructs for choroidal disorders

Technical Field

The application relates to the field of ophthalmic disease gene therapy and molecular medicine, in particular to application of a CHM gene in preparing a choroideremia therapeutic agent and a therapeutic product containing a CHM gene coding sequence.

Background

Choroideremia (OMIM 303100) is an X-linked recessive inherited degenerative chorioretinal disease with an incidence of about 50000-100000, which is manifested by progressive degeneration of Photoreceptors (PR), retinal Pigment Epithelium (RPE) and choroid (Hurk J V D et al, human modification, 1997,9 (2): 110-117). Most male patients develop night blindness symptoms before the age of ten years, and become slowly progressive visual field defects after adulthood, tubular visual field at late stage and even blindness. Female CHM carriers have no clinical manifestations or only slight fundus changes in the early stages, but may experience night vision deterioration in the late years. There are studies reporting that expression of REP1 protein after transfection of type 2 AAV vector carrying CHM gene into isolated human cells and retina of CHM model mice is accompanied by restoration of visual function, but the effect is limited. At present, most of the clinical treatments mainly include observation and complications, and no effective treatment method exists. Currently, some gene therapy drugs have been used for treating diseases caused by gene mutation or gene damage. However, there is still a lack of genetic drugs in the art that can effectively treat choroidal disorders.

Disclosure of Invention

In order to provide a drug without choroidal disease with better expected clinical effect, the present application provides a nucleic acid molecule capable of expressing CHM protein in eukaryotic cells and a viral particle comprising the same. The nucleic acid molecules and virus particles have significantly higher expression efficiency than the prior art at the same dose and successfully achieve a significant reversal of choroidal free disease at the animal level.

Specifically, the present application provides:

1. an engineered nucleic acid molecule that expresses a CHM protein (CHM Rab escort protein) or fragment thereof in a eukaryotic cell, said nucleic acid molecule comprising a coding sequence encoding a sequence as set forth in SEQ ID NO:10, and the amino acid sequence shown as SEQ ID NO:10, or a conservative substitution variant of an amino acid sequence as set forth in SEQ ID NO:10 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) or more than 85%.

2. The engineered nucleic acid molecule of item 1, wherein the coding sequence comprises the sequence set forth in SEQ ID NO:4 or a polynucleotide sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto. In some embodiments, the nucleic acid molecule may be chemically modified. In some embodiments, one or more nucleotides in the nucleic acid molecule are replaced with other nucleotides having the same function in the transcription and/or translation process, including, but not limited to, exchanging one or more of T (thymine) with U (uracil), replacing one or more of G (guanine) with I (creatinine), and the like.

3. The engineered nucleic acid molecule of item 1 or 2, in some embodiments, the nucleic acid molecule is an mRNA molecule or is a DNA molecule encoding an mRNA molecule. In some embodiments, the 5 'end side of the coding sequence further comprises a 5' utr sequence.

4. The engineered nucleic acid molecule of item 3, wherein the 5' utr sequence comprises the sequence set forth in SEQ ID NO:7 and/or SEQ ID NO:8 or a polynucleotide sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto. In some embodiments, the 5' utr sequence comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the 5' utr sequence comprises the sequence set forth in SEQ ID NO:8, and a polynucleotide sequence shown in SEQ ID NO. In some embodiments, the 5' utr sequence comprises the sequence set forth in SEQ ID NO:7, and the polynucleotide sequence shown in SEQ ID NO:8, and a fragment of a polynucleotide sequence linked to said SEQ ID NO:7 and a polynucleotide sequence as set forth in SEQ ID NO:8, and a short peptide sequence of the polynucleotide sequence shown in FIG. 8. In some embodiments, the short peptide is a GS linker.

5. The engineered nucleic acid molecule of item 3, wherein the 5' utr sequence comprises the sequence set forth in SEQ ID NO:9 or a polynucleotide sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto.

6. The engineered nucleic acid molecule of any one of claims 1-5, further comprising a Kozak sequence.

7. The engineered nucleic acid molecule of item 6, wherein the Kozak sequence is set forth in SEQ ID NO: shown at 5.

8. The engineered nucleic acid molecule of any one of claims 1-7, which is a DNA molecule, and further comprising an HGHpA tailing signal sequence and/or a WPRE sequence. In some embodiments, the DNA molecule further comprises a coding sequence for a Poly (a) tail and/or a WPRE sequence.

9. The engineered nucleic acid molecule of item 8, wherein the HGHpA tailing signal sequence is as set forth in SEQ ID NO:2, the WPRE sequence is shown as SEQ ID NO: shown at 6.

In some embodiments, the DNA molecule further comprises a promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the promoter comprises the amino acid sequence set forth in SEQ ID NO:1, and a polynucleotide sequence shown in seq id no.

10. The engineered nucleic acid molecule of item 1, comprising the sequence set forth in SEQ ID NO:11 to 13 or a polynucleotide sequence having more than 85% (e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto.

In some embodiments the nucleic acid molecule has the ability to insert into the host genome, e.g., comprises a transposon sequence.

In addition, the application also provides:

11. an engineered nucleic acid molecule that can express a CHM protein or fragment thereof in a eukaryotic cell, comprising, in order from 5 'to 3' end, a CMV promoter, a 5'utr, a Kozak sequence, and a CHM coding sequence, wherein the 5' utr sequence comprises the amino acid sequence as set forth in SEQ ID NO:7 and/or SEQ ID NO:8, or a polynucleotide sequence as set forth in SEQ ID NO:7 and/or SEQ ID NO:8 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) of the sequence identity.

12. The engineered nucleic acid molecule of item 11, the 5' utr sequence comprising the sequence set forth in SEQ ID NO:9 or a polynucleotide sequence as set forth in SEQ ID NO:9 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) of the sequence identity.

13. The engineered nucleic acid molecule of item 11 or 12, which is a DNA molecule, and further comprising an HGHpA tailing signal sequence and/or a WPRE sequence.

14. The engineered nucleic acid molecule of item 13, wherein the HGHpA tailing signal sequence is as set forth in SEQ ID NO:2 or a sequence as set forth in SEQ ID NO:2 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) and the WPRE sequence as set forth in SEQ ID NO:6 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%).

15. The nucleic acid molecule of any one of claims 11 to 14, wherein the CMV promoter sequence is set forth in SEQ ID NO:1 or a sequence as set forth in SEQ ID NO:1 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%). 16. The engineered nucleic acid molecule of any one of claims 11 to 15, wherein an intron sequence comprising the sequence set forth in SEQ ID NO:14 or an intron sequence as set forth in SEQ ID NO:14 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%).

17. The engineered nucleic acid molecule of item 16 comprising on the 5' side of the coding sequence an amino acid sequence as set forth in SEQ ID NO:15 to 17 or a polynucleotide sequence as set forth in any one of SEQ ID NOs: 15 to 17, wherein the polynucleotide sequence has more than 85% (e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity.

18. The engineered nucleic acid molecule of any one of claims 11 to 17, wherein the coding sequence encodes a sequence as set forth in SEQ ID NO:10, or an amino acid sequence as set forth in SEQ ID NO:10, or a conservative substitution variant of an amino acid sequence as set forth in SEQ ID NO:10 (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) or more than 85%.

19. The engineered nucleic acid molecule of item 18, wherein the coding sequence comprises the sequence set forth in SEQ ID NO:4 or a polynucleotide sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto.

20. The engineered nucleic acid molecule of any one of claims 11 to 18, comprising the amino acid sequence set forth in SEQ ID NO:11 to 13 or a polynucleotide sequence having more than 85% (e.g. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) sequence identity thereto.

21. The engineered nucleic acid molecule of any one of claims 1-20, which is an AAV core expression plasmid.

22. A viral particle comprising the engineered nucleic acid molecule of any one of claims 1-21.

23. The viral particle of item 22, which is an AAV viral particle.

24. The viral particle of item 23, wherein the capsid protein serotype is AAV8.

25. Use of the engineered nucleic acid molecule according to any one of claims 1-21 or the viral particle according to any one of claims 12 to 14 for the preparation of a medicament for the treatment of choroidal space.

26. The use according to item 25, wherein the medicament is administered through the subretinal space to treat choroidal space.

The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the scope of the technical idea of the present application, a number of simple variants of the technical solution of the present application are possible, including that the individual technical features are combined in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed in the present application, all falling within the scope of protection of the present application. Aspects and embodiments of the present application described herein include aspects and embodiments that "comprise," consist of, "and" consist essentially of … ….

Drawings

FIG. 1 is a plasmid map of expression vector 1.

FIG. 2 is a plasmid map of expression vector 2.

FIG. 3 is a plasmid map of expression vector 3.

FIG. 4 is a plasmid map of expression vector 4.

FIG. 5 is a plasmid map of expression vector 5.

FIG. 6 is a statistical chart showing the relative expression levels (A) of mRNA and (B) of CHM gene after infection of HEK293T cells with AAV virus particles packed with the expression vectors 1 to 5, respectively.

FIG. 7 shows a comparison of the infection efficiency of AAV2 and AAV8 in vivo mouse retinal cells

Fig. 8 is a view of the apparent dynamic response statistics and the amplitude statistics of ERG (electroretinogram) of the subject.

Fig. 9 is a plot of HE staining of the retinas of mice in each group.

Detailed description of the preferred embodiments

The present application relates to a nucleic acid molecule capable of expressing CHM protein in eukaryotic cells, a viral particle comprising said nucleic acid molecule, and the use of said nucleic acid molecule and viral particle for the preparation of a medicament for the treatment of choroidal disorders. The nucleic acid molecules and the virus particles are modified to have remarkably higher expression efficiency, can treat the choroideremia with lower dosage, and successfully realize the reversion of the CHM deficiency related diseases at animal level.

Definition of the definition

For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the technology belongs. All technical and patent disclosures cited herein are incorporated herein by reference in their entirety.

As used herein, the term "CHM" or "CHM protein" is used to refer to a protein encoded by the CHM Rab guard protein (Rab escort protein) gene. The gene codes for the A component of the RAB geranylgeranyl transferase holoenzyme. In some embodiments, the CHM protein is a human CHM protein. An exemplary CHM protein is encoded by a gene with a gene ID of 1121 in the NCBI database. In some embodiments, the CHM or CHM protein comprises the amino acid sequence set forth in SEQ ID NO:10, and a polypeptide having the amino acid sequence shown in FIG. 10.

As used herein, a percentage of "identity," e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% identity, refers to a degree of similarity between amino acid sequences or between nucleotide sequences, as determined by sequence alignment, of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%. For example, by introducing gaps or the like, it is possible to determine the ratio of the number of positions having the same base or amino acid residue to the total number of positions after two sequences have the same residue at as many positions as possible. The percentage of "identity" may be determined using software programs known in the art. Preferably, the alignment is performed using default parameters. One preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these programs can be found at the following internet addresses: ncbi.nlm.nih.gov/cgi-bin/BLAST.

As used herein, a "variant" has at least one amino acid difference, e.g., at least one amino acid addition, insertion, deletion, or substitution, relative to a reference amino acid sequence. For example, the amino acid substitutions may be conservative amino acid substitutions, i.e., the original corresponding amino acid is substituted with an amino acid having similar properties. "conservative substitutions" may be polar versus polar amino acids, such as glycine (G, gly), serine (S, ser), threonine (T, thr), tyrosine (Y, tyr), cysteine (C, cys), asparagine (N, asn), and glutamine (Q, gin); nonpolar to nonpolar amino acids such as alanine (a, ala), valine (V, val), tryptophan (W, trp), leucine (L, leu), proline (P, pro), methionine (M, met), phenylalanine (F, phe); acidic versus acidic amino acids, such as aspartic acid (D, asp), glutamic acid (E, glu); basic para-basic amino acids such as arginine (R, arg), histidine (H, his), lysine (K, lys); charged amino acids versus charged amino acids such as aspartic acid (D, asp), glutamic acid (E, glu), histidine (H, his), lysine (K, lys), and arginine (R, arg)); hydrophobic versus hydrophobic amino acids such as alanine (a, ala), leucine (L, leu), isoleucine (I, ile), valine (V, val), proline (P, pro), phenylalanine (F, phe), tryptophan (W, trp) and methionine (M, met). In some other embodiments, the variants may also comprise non-conservative substitutions. In some embodiments, the "variant" of the amino acid sequence may have at least about 90%,95%,96%,97%,98%,99% sequence identity to the amino acid sequence. A "variant" of the amino acid sequence may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%,97%,98%,99% or 100% or a range of any two of the foregoing values, as compared to the amino acid sequence. As used herein, a "conservatively substituted variant" of a protein, polypeptide, or amino acid sequence refers to a protein in which one or more amino acid residues are substituted with amino acids, without altering the overall conformation and function of the protein or enzyme, including but not limited to the substitution of amino acids in the amino acid sequence of the parent protein in the manner described for the aforementioned "conservative substitution". Thus, the similarity of two proteins or amino acid sequences of similar function may be different. For example, 70% to 99% similarity (identity) based on the megasign algorithm. "conservatively substituted variants" also include polypeptides or enzymes having more than 60% amino acid identity, more preferably more than 75%, and most preferably more than 85% and even more preferably more than 90% as determined by BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

As used herein, "amino acid" refers to any monomeric unit that may be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 naturally or genetically encoded α -amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). Amino acids also include unnatural amino acids, modified amino acids (e.g., with modified side chains and/or backbones), and amino acid analogs. To further illustrate, the amino acid is typically an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, mercapto, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxy, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, phospho, phosphonyl, phosphine, heterocycle, ketene, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photosensitive cross-linker, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids containing new functional groups, amino acids that interact covalently or non-covalently with other molecules, photolabile (photocaged) and/or photoisomerisable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogues, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids comprising carbon linked sugars, redox active amino acids, amino acids comprising amino thio acids, and amino acids comprising one or more toxic moieties. Amino acids described herein include, but are not limited to: 20 natural amino acids and 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminoalanine (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or piperidineic acid (4 Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminomethacrylic acid (bAib), 2-aminopimelic acid (Apm), 2, 4-diaminobutyric acid (Dbu), methamphetamine (Des), 2' -diaminopimelic acid (Dpm), 2, 3-diaminopropane sulfonic acid (Dpr), ethylglycine (EtGly), N-ethylaspartic acid (EtAsn), hydroxylysine (Hyl), isolysin (aHyl), 3-hydroxyproline (3 Hyp), 4-hydroxyproline (4 Hyp), isodesmin (Ide), isoleucine (NIle), N-methylglycine or myo (MeGly), N-methylisoleucine (MeN-methyl-N, 6-methylvaline (Mevaline), norvaline (Mevaline) and norvaline (Val). Thus, in some embodiments, the amino acid mutation at the site comprises a substitution mutation that converts to any of the 20 natural amino acids described above and the unnatural amino acids described above, after the mutation. In some embodiments, the amino acid mutation comprises a substitution mutation that converts to any one of the amino acids selected from the group consisting of: G. a, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H, aad, bAad, bAla, abu, 4Abu, acp, ahe, aib, bAib, apm, dbu, des, dpm, dpr, etGly, etAsn, hyl, aHyl, 3Hyp, 4Hyp, ide, aIle, meGly, meIle, meLys, meVal, nva, nle, and Orm.

In the context of the present invention, the terms "DNA" and "RNA" refer to single-or double-stranded DNA or RNA molecules. Unless otherwise indicated, the terms "DNA" and "DNA molecule" refer to double-stranded DNA molecules consisting of A, C, G and/or T nucleotides, while the terms "RNA" and "RNA molecule" refer to single-stranded RNA molecules consisting of A, C, G and/or U nucleotides. In this context, the A, C, G, T and U nucleotides refer to nucleotides comprising adenine, guanine, cytosine, thymine and uracil as the respective nitrogenous bases.

RNA molecules include coding RNA (coding RNA) or non-coding RNA (ncRNA), such as Pre-mRNA, mature mRNA or long non-coding RNA (lncRNA).

As used herein, the "hybrid molecule of DNA and RNA" is a molecule comprising a polynucleotide sequence consisting of deoxyribonucleotides and ribonucleotides. The hybrid molecule of DNA and RNA can be obtained by:

replacing one or more deoxyribonucleotides in the DNA with ribonucleotides;

replacing one or more ribonucleotides in the RNA with deoxyribonucleotides; or (b)

Deoxyribonucleotides and ribonucleotides are synthesized from the head by biological or chemical synthesis. It should be noted that the manner of obtaining the hybrid molecule of DNA and RNA is not limited to the above manner, and the hybrid molecule of DNA and RNA obtained by any manner falls within the category of "hybrid molecule of DNA and RNA" as defined herein.

As used herein, if two nucleic acid molecules are described as having "identical genetic information," it is meant that the two nucleic acid molecules are complementary or comprise identical base sequences, or that one or more thymines in the base sequence of one of the nucleic acid molecules are converted to uracil, resulting in a nucleic acid molecule that is identical to the base sequence of the other nucleic acid molecule. Thus, any two of the DNA, RNA, and hybrid molecules of DNA and RNA may have the same genetic information. Wherein the term "base sequence" refers to the order of arrangement of bases in a polynucleotide molecule. It will be appreciated by those skilled in the art that unless otherwise indicated, a base sequence or polynucleotide sequence as described herein may be referred to as "T" when used to describe a DNA sequence, but that "T" will be replaced by "U" (uracil) when the base sequence or polynucleotide sequence is used to describe RNA (e.g., mRNA). Thus, any DNA disclosed by a particular sequence number (SEQ ID NO) herein also discloses an RNA (e.g., mRNA or Poly (a) tail) sequence complementary or corresponding to the DNA, wherein each "T" of the DNA sequence is substituted with a "U". As used herein, the term "about" refers to the usual error range for individual values as readily known to those of skill in the art. References to "about" a value or parameter include (and describe) embodiments directed to the value or parameter itself. As used herein, when the term "about" precedes a numerical value, it means within 10% of the value above or below. For example, "about 100" encompasses 90 and 110.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise herein, 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.

Nucleic acid molecules

A first aspect of the present application relates to an engineered nucleic acid molecule capable of expressing a CHM protein or fragment thereof in a eukaryotic cell, said nucleic acid molecule comprising a coding sequence encoding a sequence as set forth in SEQ ID NO:10, and the amino acid sequence shown as SEQ ID NO:10, or a conservative substitution variant of an amino acid sequence as set forth in SEQ ID NO:10, and the amino acid sequence has more than 85% of sequence identity. Among these, there are two cases of "an engineered nucleic acid molecule capable of expressing a CHM protein or a fragment thereof in a eukaryotic cell", one of which is inserted into the genome of a eukaryotic cell by transposition or homologous recombination, whereby the CHM protein is expressed by using the essential elements of the genome itself at the insertion site, and the other of which is comprised of the essential elements which are useful for expressing the CHM protein in a eukaryotic cell, i.e., which itself, after entering a eukaryotic cell, is translated or post-transcriptionally translated to produce the CHM protein or a fragment thereof.

Thus, in some embodiments, the engineered nucleic acid molecule is an engineered DNA molecule. In some embodiments, the engineered DNA molecule may be transcribed into mRNA. In some embodiments, the engineered DNA molecule comprises one or more coding sequences of elements useful for initiating or regulating expression or translation of the CHM protein or fragment thereof after transcription, including but not limited to 5'utr, 3' utr, poly (a) tail (or tailing signal), WPRE, and the like. In the present application, when referring to translational regulatory elements of the 5'UTR, 3' UTR, poly (A) tail, tailing signal, WPRE, etc., it is an RNA sequence in the context of mRNA and a DNA sequence encoding or transcribing said RNA sequence in DNA. In some embodiments, the tailing signal is an HGHpA tailing signal. In some embodiments the tailing signal sequence comprises the sequence as set forth in SEQ ID NO:2, and a polynucleotide sequence shown in seq id no. In some embodiments, the polynucleotide sequence of the tailing signal is as set forth in SEQ ID NO: 2. In some embodiments, the WPRE sequence is as set forth in SEQ ID NO: shown at 6.

In some embodiments, the engineered DNA molecule comprises a coding sequence for at least one untranslated region (UTR). In some embodiments, the engineered DNA molecule comprises a coding sequence for at least a 5' utr and a coding sequence for the CHM protein or fragment thereof. In some embodiments, the engineered DNA molecule comprises at least the coding sequence of the 5'utr, the coding sequence of the CHM protein or fragment thereof, the coding sequence of the 3' utr, the tailing signal (or DNA sequence corresponding to the Ploy (a) tail sequence) in sequence from 5 'to 3', and may further comprise a start codon (5 'end) and a stop codon (3' end), respectively, at both ends of the coding sequence of the CHM protein or fragment thereof, which are the first three nucleotides and the last three nucleotides, respectively, of the mRNA molecule that can be translated. The 5' UTR typically comprises at least one Ribosome Binding Site (RBS), such as the Shine-Dalgarno sequence in prokaryotes, or at least one translation initiation site, such as the Kozak sequence in eukaryotes. However, in the present application, when referring to "5'UTR sequence" it is meant that the partial sequence of the 5' UTR does not contain a ribosome binding site or translation initiation site. RBS promotes efficient and accurate translation of mRNA molecules by recruiting ribosomes at the beginning of translation. The activity of a given RBS or translation initiation site can be optimized by varying its length and sequence as well as distance from the initiation codon. Alternatively or optionally, the 5' utr comprises an internal ribosome entry site or IRES. The 3' utr may comprise one or more regulatory sequences, such as binding sites for amino acid sequences that enhance stability of the mRNA molecule, binding sites for regulatory RNA molecules (e.g., miRNA molecules), and/or signal sequences involved in intracellular transport of the mRNA molecule.

On the basis of the foregoing embodiments, in some embodiments, the engineered nucleic acid molecule further comprises one or more additional regulatory sequences, such as binding sites for amino acid sequences that enhance stability of the mRNA molecule, binding sites for amino acid sequences that enhance translation of the mRNA molecule, regulatory elements (e.g., riboswitches), and/or nucleotide sequences that positively affect translation initiation. Furthermore, within the 5' utr, there is preferably no functional upstream open reading frame, an out-of-frame upstream translation initiation site, an out-of-frame upstream initiation codon, and/or a nucleotide sequence that produces a secondary structure that reduces or prevents translation. The presence of such nucleotide sequences in the 5' UTR can negatively impact translation.

Different 5' UTRs may bring about different expression effects for the same coding sequence. Optimally, when the nucleic acid molecules of the present application comprise a sequence as set forth in SEQ ID NO:7 and/or SEQ ID NO:8, and a significantly higher level of expression of the CHM protein or fragment thereof can be obtained, and a restoration of retinal function at the animal level can be successfully achieved by expressing the CHM protein or fragment thereof. In some embodiments, the nucleic acid molecules of the present application comprise a sequence as set forth in SEQ ID NO:9, and a 5' UTR coding sequence shown in FIG. 9. In some embodiments, the 5'utr further comprises a coding sequence for a Kozak sequence at the 3' end. In some embodiments, the coding sequence of the Kozak sequence of the present application is optimized to comprise the sequence set forth in SEQ ID NO:5, and a nucleotide sequence shown in SEQ ID NO.

The coding sequence comprises codons that can be translated into an amino acid sequence. The coding sequence may comprise all naturally occurring codons encoding amino acids, or may comprise some or all of artificial codons. In some embodiments, the portion or all of the codons are codon optimized. In some embodiments, the partial or all codons encode an unnatural amino acid. In some embodiments, the coding sequence comprises the sequence set forth in SEQ ID NO:4 or a polynucleotide sequence having 85% or more sequence identity thereto.

In some embodiments, the engineered DNA molecule further comprises structural elements on the 5' side of the coding sequence of the engineered nucleic acid molecule necessary to initiate or regulate transcription of the RNA, which structural elements are known in the art. In some embodiments, the structural element comprises at least a promoter. Promoters and their sequences are known in the art and include weak promoters, medium strength promoters, strong promoters, mini promoters or core promoters, and the like. In some specific embodiments, the promoter is a strong promoter. In some embodiments, the promoter may initiate transcription of the coding sequence of the CHM protein or fragment thereof in eukaryotic cells. The "promoter" comprises at least one transcription recognition site followed by a transcription factor binding site. The recognition and binding sites may interact with amino acid sequences that mediate or regulate transcription. The binding site is closer to the aforementioned coding sequence than the recognition site. The binding site may be, for example, a Pribnow box in prokaryotes or a TATA box in eukaryotes. For example, in some embodiments, when the Pribnow box is used, the transcription recognition site may be located about 35bp upstream of the transcription start site, and the transcription factor binding site may be located about 10bp upstream of the transcription start site. In some embodiments, the promoter comprises AT least one additional regulatory element, such as an AT-rich upstream element located about 40 and/or 60 nucleotides before the transcription initiation site, and/or an additional regulatory element located between the recognition site and the binding site that enhances promoter activity. In some embodiments, the promoter is a strong promoter, i.e., the promoter comprises sequences that promote transcription of the aforementioned RNA coding sequences. Strong promoters are known to those skilled in the art, and CMV promoters, EF1A promoters, CAG promoters, CBh promoters, SFFV promoters, etc., or can be identified or synthesized by routine laboratory procedures. In some embodiments, the promoter is preceded by additional regulatory elements, such as enhancers that facilitate transcription of the aforementioned RNA coding sequences, contained in a DNA plasmid.

In some embodiments, the engineered DNA molecule may be expressed not only in eukaryotic cells but also replicated in prokaryotic cells. The engineered DNA molecule thus comprises, in addition to the coding sequence encoding the CHM protein or fragment thereof, genetic manipulation or regulatory elements for replication and/or expression in prokaryotic and/or eukaryotic cells.

Replication or efficient replication of the engineered DNA molecule in a cell the necessary structural elements are known in the art and include, for example, an Origin of Replication (ORI). In some embodiments, the engineered DNA molecule still further comprises a marker gene or fragment thereof and/or a reporter gene or fragment thereof, and a unique restriction enzyme site that allows for insertion of DNA elements, preferably in the form of a Multiple Cloning Site (MCS). The marker gene facilitates identification of cells containing a plasmid comprising the marker gene, which may be selected from, for example, antibiotic resistance genes. Each restriction enzyme site in the MCS may be specifically recognized by a different restriction enzyme.

In some embodiments, the engineered DNA molecule is a DNA plasmid. As used herein, the term "DNA plasmid" refers to a plasmid consisting of a double stranded DNA molecule. In some embodiments, the "plasmid" is a circular DNA molecule. In some embodiments, the "plasmid" may also encompass a linear DNA molecule. In particular, the term "plasmid" also covers molecules obtained by linearizing a circular plasmid by, for example, cleaving the circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule, as well as linear molecules that can replicate in prokaryotes. Plasmids can replicate, i.e., amplify in cells independent of the genomic genetic information stored by the prokaryotic pseudonucleus or the pseudonucleus, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. Preferably, the DNA plasmid according to the invention is a medium-or high-copy plasmid, more preferably a high-copy plasmid. Examples of such high copy plasmids are such vectors: it is based on pUC, pTZ plasmid or any other plasmid containing ORI supporting high copies of the plasmid (e.g.pMB1, pCoIE1) etc.

In some embodiments, the nucleic acid molecule is a viral vector plasmid. As used herein, the term "viral vector" plasmid refers to any plasmid that contains or can be inserted into a coding sequence for a protein gene of interest (e.g., a coding sequence for the CHM protein or fragment thereof of the present application), and that can be used for viral packaging, including, but not limited to, AAV core expression plasmids, lentiviral transfer plasmids, and the like.

In some embodiments, the nucleic acid molecule is an adeno-associated virus (AAV) core expression plasmid, i.e., the nucleic acid molecule further comprises an Inverted Terminal Repeat (ITR) of AAV. In some embodiments, the nucleic acid molecule is an AAV genomic nucleic acid or portion thereof. In some embodiments, the eukaryotic cell is a HEK-293 cell. In some embodiments, the eukaryotic cell is a photoreceptor cell or a Retinal Pigment Epithelium (RPE) cell. As used herein, the term "photoreceptor cell" may be used to refer to any one or more of a rod cell, a cone cell, and a photosensitive ganglion cell. In some embodiments, the AAV is an AAV in which the capsid protein (CAP) is derived from serotype 2 or 8 (and AAV2 or AAV 8). In some embodiments, the AAV is one in which the capsid protein is derived from AAV8 and its gene (REP) that regulates AAV replication is derived from AAV2.

In some embodiments, the engineered DNA molecule is a DNA molecule or fragment thereof that constitutes a prokaryotic pseudocore or a nucleoid, or a DNA molecule or fragment thereof that constitutes a eukaryotic genome, i.e., the coding sequence of the aforementioned CHM protein or fragment thereof, or the complement thereof, is replicable with the prokaryotic genome.

In some embodiments, the engineered nucleic acid molecule is an engineered RNA molecule that comprises the coding sequence of the CHM protein or fragment thereof described above, and can express the CHM protein or fragment thereof in eukaryotic cells. In some embodiments, the engineered nucleic acid molecule is an engineered RNA molecule and comprises a complement of the coding sequence of the CHM protein or fragment thereof described above. In some embodiments, the engineered RNA molecule is an mRNA molecule obtained by transcription of the engineered DNA molecule described above. In some embodiments, the engineered RNA molecule is identical to or comprises an mRNA molecule transcribed from the engineered DNA molecule described above.

In some embodiments, the engineered nucleic acid molecule may also be a hybrid molecule of DNA and RNA. The hybrid molecule of DNA and RNA contains the same genetic information as the engineered RNA molecule or engineered DNA molecule described above.

Virus particles, nanoparticles, cells and uses

The second aspect of the present application also provides a liposome, nanoparticle, or viral particle comprising the engineered nucleic acid molecule of the foregoing first aspect. In some embodiments, the viral particle is a lentivirus or other retrovirus, adenovirus, AAV, or baculovirus. In some embodiments, the viral particle is an AAV virus having a capsid protein serotype of AAV8 or AAV 2.

The present application also provides a cell comprising the engineered nucleic acid molecule of the first aspect described above. In some embodiments, the cells are prokaryotic cells, which may be used for in vitro amplification of the aforementioned engineered DNA molecules. In some embodiments, the cells are eukaryotic cells, which may be used for packaging of the viral particles. Thus in some embodiments, the eukaryotic cell is a mammalian cell, such as a HEK-293 cell. In some embodiments, the eukaryotic cell is a photoreceptor cell or a retinal pigment epithelial cell, wherein the gene encoding the CHM protein in the genome of the cell has a pathogenic mutation that results in loss or attenuation of CHM protein function. In some embodiments, the eukaryotic cell is a stem cell or progenitor cell that can differentiate into a photoreceptor cell or a retinal pigment epithelial cell, which is not an animal embryonic stem cell, and is not a stem cell isolated or obtained using a human embryo within 14 days of fertilization that has not undergone in vivo development.

Methods of treating choroidal disorders are also provided. In some embodiments, the method comprises contacting the engineered nucleic acid molecule according to the foregoing first aspect or the liposome, nanoparticle, or viral particle of the second aspect with a photoreceptor cell and/or retinal pigment epithelial cell of a patient. The present application also provides the use of an engineered nucleic acid molecule according to the first aspect or a liposome, nanoparticle, or viral particle of the second aspect, or the cell comprising an engineered nucleic acid molecule of the first aspect, as described above, for the manufacture of a medicament for the treatment of choroidal space. In some embodiments, the method or use comprises administering to a patient or subject in need thereof an engineered nucleic acid molecule according to the first aspect or a liposome, nanoparticle, or viral particle of the second aspect described above by means of subretinal space administration. In some embodiments, the method or use comprises administering to a patient or subject in need thereof a cell comprising the engineered nucleic acid molecule of the first aspect described above and inducing differentiation thereof into a photoreceptor cell and/or a retinal pigment epithelial cell. In some embodiments, the method or use comprises administering to a patient or subject in need thereof a susceptor cell and/or retinal pigment epithelial cell obtained from the differentiation of a cell comprising the engineered nucleic acid molecule of the first aspect described above, and allowing it to colonize the retina.

It is to be understood that this application encompasses the various aspects, embodiments, and combinations of the aspects and/or embodiments described herein. The above description and the examples that follow are intended to illustrate and not limit the scope of the present application. Within the scope of the technical idea of the present application, a number of simple variants of the technical solution of the present application are possible, including that the individual technical features are combined in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed in the present application, all falling within the scope of protection of the present application.

The practice of this application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology. The prior art documents describe said conventional art.

Examples

The practice of this application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology.

EXAMPLE 1 construction of rAAV-CMV-hCHM recombinant expression vector

Studies in this application have shown that the use of different CHM expression vectors can produce different therapeutic effects on choroidal-free conditions. In order to find the optimal expression vector, three generations of AAV recombinant expression vectors are designed, wherein the first generation is the expression vector 1, the second generation is the expression vector 2, and the third generation is the expression vectors 3-5.

The structure of expression vectors 1 and 2 (without 5' UTR, using HGHpA tailing signal sequence) is shown in FIGS. 1 and 2, respectively. Wherein the sequence of the CMV promoter is shown as SEQ ID No. 1; the sequence of HGHpA is shown as SEQ ID No. 2. The difference between the expression vectors 1 and 2 is that different CHM coding sequences are used, wherein the CHMwt sequence used by the expression vector 1 is shown as SEQ ID NO. 3, and the CHM-sgopt used by the expression vector 2 is a codon optimized CHM gene, and the sequence is shown as SEQ ID NO. 4.

The structures of the expression vectors 3-5 are shown in the figures 3-5, and are rAAV-CMV-Intron-5' UTR-Kozak-CHMsgopt-WPRE-HGHpA structures, wherein CMV refers to CMV promoter. In eukaryotic expression systems, kozak sequences, shown in SEQ ID NO. 5, generally increase the efficiency of transcription and translation; the DNA coding sequence of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is shown in SEQ ID NO. 6.

Research shows that the reasonable design of the 5 'untranslated region (5' UTR) can improve the expression level of exogenous proteins. Expression vectors 3-5 differ in the 5' UTR, and we designed multiple 5' UTR sequences in experiments, and found that 5' UTR-2 (SEQ ID NO: 7) and 5' UTR-3 (SEQ ID NO: 8) have significantly better effects than other non-optimized 5' UTRs, the 5' UTR of expression vector 3 is 5' UTR-2, the 5' UTR of expression vector 4 is 5' UTR-3, the 5' UTR of expression vector 5 is concatenated 5' UTR-2 and 5' UTR-3, and the two sequences are linked by linker, namely 5' UTR-2-3 (SEQ ID NO: 9).

EXAMPLE 2 preparation and purification of rAAV Virus

The present invention uses standard three plasmids (AAV core expression plasmid pAAV-hCHM (laboratory construction) carrying CHM gene), pAAV helper plasmid (purchased from Agilent corporation), pAAV-RC2/8 serotype plasmid or pAAV-RC2/2 serotype plasmid (purchased from Agilent corporation), wherein RC2/2 represents both the Rep gene and Cap gene from AAV2 serotype; RC2/8 represents the Rep gene from AAV2 and Cap gene from AAV8 serotype); the rAAV-hCHM virus produced by HEK-293 cell packaging was transfected with the three plasmids and purified by density gradient ultracentrifugation. The specific process comprises the following steps: the core expression plasmid pAAV-hCHM, which constructs the hCHM gene expression cassette synthetically, was designed and produced in bacterial cells. Plasmids have the various necessary regulatory elements (AAV 2-ITR, CMV promoter, CDS sequence of hCHM, SV40 polyA). For detection, we also constructed a corresponding core expression plasmid carrying a Green Fluorescent Protein (GFP) reporter gene, i.e. the sequence encoding the P2A self-cleaving polypeptide and the Green Fluorescent Protein (GFP) reporter gene was ligated at the C-terminus of the hCHM gene.

HEK-293 cells (providing various factors for rAAV production in HEK-293 adherent cells) were expanded on petri dishes and transfected with three plasmids.

Transfected cells were cultured for a period of time to package AAV, lysed and purified by density gradient ultracentrifugation. Recombinant adeno-associated virus rAAV-hCBM is obtained, the promoter is Cytomegalovirus (CMV) promoter, and the marker gene is EGFP. The purified rAAV/hCHM was assayed by qPCR and had a titer of about 2E+12vg/mL. The purified product rAAV/hCBM was stored at-80℃and used for preclinical animal in vivo studies.

EXAMPLE 3 comparison of the characteristics of AAV 2-type and AAV 8-type recombinant expression vectors for retinal infection in animal experiments

AAV2 and AAV8 are two AAV serotypes that are more commonly used in gene therapy, and in order to facilitate comparison of the differences in the efficiency of infection of the retina by AAV of different serotypes in animal living beings, we used rAAV viral vectors rAAV2/2-CMV-hCHM-P2A-EGFP-HGHpA and rAAV2/8-CMV-hCHM-P2A-EGFP-HGHpA carrying the same infectious dose of the GFP reporter gene fused at the C-terminus of the CHM gene of vector 1 for testing.

We prefer the mode of subretinal space injection administration: the C57 mice were fixed on a mouse adapter and the mouse stage was adjusted to the appropriate angle under a dissecting microscope to ensure clear observation of the mouse cornea and limbus under the microscope. The needle is inserted at the position about 0.5mm behind the corneoscleral limbus, the inclined plane of the needle point is upward, the needle is inserted at the needle insertion point perpendicular to the sclera, the needle point is quickly turned to the optic nerve direction (namely, the inclined horizontal plane is 45 degrees) until the needle point reaches the subretinal space after breakthrough is felt, and virus is slowly injected into the subretinal space. Sterilizing after operation, placing the mice in a rewarming blanket for resuscitation, and loading the mice into a squirrel cage for continuous feeding after resuscitation.

Mouse retina frozen sections and microimaging: after 4 weeks of virus injection, the mice are sacrificed by breaking the vertebrae, the eyeballs are rapidly taken out and placed in PBS solution, the eyeballs are transferred into paraformaldehyde solution for fixation for about 40 minutes after a puncture is made on the corneoscleral limbus, and the structures such as cornea, crystal, sclera and the like are cut off after fixation, and retinal tissues are completely taken out. Sequentially transferring the cleaned retina into sucrose solution with gradient concentration (10%, 20% and 30%) for dehydration; embedding and freezing the slice with the thickness of 14 mu m, and adhering the slice to a microscopic glass slide after the retina tissue is trimmed to the target position; after selecting and developing, adding DAPI staining solution, and applying for 8 minutes at room temperature in a dark place; then PBS solution soaks retinal tissue, finally drops proper amount of anti-fluorescence quenching sealing tablet on retina and slowly covers cover glass, and prevents bubbles in the tissue during operation. The prepared retina sample is photographed by microscopic imaging or stored in a refrigerator at the temperature of minus 20 ℃ in a dark place.

As shown in fig. 7, AAV of serotypes 2 and 8 targeted photoreceptor cells and RPE cells well targeted using subretinal space injection into the eyeball of C57 mice, and AAV8 targeted photoreceptor cells more efficiently than AAV 2. Furthermore, at the same drug dose, AAV8 vector was significantly higher than GFP expression level of AAV2 vector, so we prefer AAV8 serotype for choroidal-free gene therapy studies.

Example 4: comparison of in vitro expression efficiency of HEK293T cells infected by different rAAV expression vector viruses

1) Infection of HEK293T cells: dividing 6-well plate into 5 experimental groups, and inoculating 5×10 respectively ⁵ HEK293T cells of (1X 10) in each well 1 day after seeding ⁶ About, 50. Mu.l (1X 10) ¹⁰ vg), expression vector 1 viral particles (group a), expression vector 2 viral particles (group B), expression vector 3 viral particles (group C), expression vector 4 viral particles (group D) and expression vector 5 viral particles (group E). After 48 hours of infection, mRNA and protein of the infected cells were extracted and analyzed separately.

2) Q-PCR detection of relative expression of CHM Gene at mRNA level: first, total RNA of cells is extracted by using Trizol kit and is synthesized into cDNA template by reverse transcription. Then, detection was performed by fluorescent quantitative PCR. Q-PCR reaction system: SYBR Green II 10 μl; 0.3 μl of each of the target fragment primers; ROX 0.4 μl; sterile water was added to 20 μl. The Q-PCR reaction procedure was: pre-denaturation at 95℃for 5min; cycling 40 times: 15s at 95℃and 1min at 60 ℃. The difference in CHM gene expression level was analyzed by a 2- ΔΔct relative quantification method.

3) Western blot detection of relative expression amount of CHM gene at protein level: different groups of HEK293T cells are separated, the culture solution is removed, the culture solution is washed once by PBS, the lysate is added for complete lysis, 12000g is centrifuged for 5 minutes, and the supernatant is taken. Protein concentration was measured by BCA method, and then loading volumes of each group were calculated as 30 μg of total protein, subjected to SDS-PAGE gel electrophoresis and Western blot detection, and subjected to gray scale quantitative analysis by Image J software.

As a result, as shown in FIG. 6, the relative expression amount of the target protein was significantly increased in group B, C, D, E compared to group A, in which the mRNA level was increased by about 3.5 times and the protein level was increased by about 4 times, both at the mRNA level and the protein level. C. The D, E group had an increase in mRNA levels of about 8-fold or more and protein levels of about 7-fold or more. Thus, the expression level of the CHM gene can be significantly increased by codon optimization and incorporation of the preferred 5' utr. After optimization, the second generation and third generation expression vectors have higher in vitro infection and expression efficiency, especially the E group (the expression vector 5 after the two 5' UTRs are connected in series), and compared with the A group (the expression vector 1), the mRNA level reaches about 11 times, and the protein level reaches about 10 times of the A group. Therefore, the expression vector 5 was selected for the subsequent animal experiments.

Example 5: verification of effectiveness of rAAV2/8-hCHM recombinant expression vector in animal experiment

The invention adopts Cre-LoxP technology to construct CHM model mice (Chm ^fl/+ Six 3-Cre) as a model for verifying the effectiveness of CHM therapeutic agents, by CHM from the laboratory ^f/+ The mice are obtained by hybridization propagation with Six3-Cre tool mice. (wherein Chu ^f/+ Mice were constructed by Jiangsu Jiyaokang biotechnology Co., ltd, and Six3-Cre tool mice were derived from Jakson laboratories, USA, strain number: 019755). All animal experiments are carried out in clean environment, the animal experiments are carried out by national standard feed and filtered sterile water, the temperature and the humidity are constant, the illumination intensity is 18lux, and 12h/12h day/night circulation alternate illumination is carried out.

The CHM expression vector viral particles prepared from expression vector 5 are administered topically by intraocular injection, i.e., subretinal or vitreous cavity. In the invention, 1 mul of recombinant virus preparation is injected into the eye of a CHM model mouse with P30+/-3 through a subretinal cavity, so that the retina of the mouse expresses human CHM protein, thereby realizing the recovery of the retina structure and visual function. The method comprises the following specific steps: after the mice are filled with the compound tobican eye drops to disperse pupils, general anesthesia is carried out by using 5% chloral hydrate, under the direct vision of an anatomic microscope, a self-assembled microinjector and a 32G insulin nuo and needle head are used for obliquely injecting the mice to a vitreous cavity 1mm behind a corneoscleral limbus, taking care of avoiding iris and crystalline lens, slowly pushing and injecting 1 mul dose, and stopping the injection for 2 minutes. After operation, levofloxacin eye ointment is applied for three continuous days, once a day, so as to reduce inflammatory reaction and prevent infection.

4 weeks after treatment, CHM-KO mice were examined for restoration of retinal function using ERG. The method comprises the following specific steps:

ERG reaction: overnight in dark environment (dark adaptation condition)Or after continuous stimulation for 10 minutes under the system background light (bright adaptation condition), compound topiramate eyedrops are dropped on the surfaces of the eyes of the mice to carry out mydriasis. Subsequently, 5% chloral hydrate was injected intraperitoneally at a dose of 0.01mL/g to anesthetize the mice, and external use of oxybuprocaine hydrochloride eye drops was used to perform ocular surface anesthesia after anesthesia. After the mouse enters an anesthetic state, the electrode is connected, the recording electrode is vertically and slightly contacted with the central top end of the cornea of the mouse, and the reference electrode and the grounding electrode are respectively inserted into the subcutaneous part and the tail part between two ears of the mouse. All manipulations were performed under dark red light to ensure the dark adaptation state of the mice. Measurements were performed using a reterner 4.0 eye electrophysiological vision system, sequentially increasing light intensity stimulation and recording retinal integrated potential response. The a-wave amplitude is the potential difference from the baseline to the a-wave trough, and the b-wave amplitude is the potential difference from the a-wave trough to the b-wave trough. The results are shown in FIG. 8, where Chm after 4 weeks of treatment ^fl/+ The a and b wave amplitudes of the Six3-Cre mouse ERG are obviously larger than those of untreated subjects under the conditions of medium and high light intensity and even partial low light intensity. Overall, the a, b wave amplitudes recovered approximately to the 1/3 level of the littermate control mice under the highest light intensity conditions.

Retinal results were detected by HE staining: mice were sacrificed by cervical dislocation after deep anesthesia, eyeballs were removed, rinsed with PBS, and 4% paraformaldehyde fixed for 24 hours. After conventional gradient dehydration, paraffin-embedded sections were cut along the sagittal axis parallel to the optic nerve, 4 μm thick, immersed in xylene at 60℃for 10-5 minutes. HE staining and observing under an optical microscope after sealing. As a result, as shown in FIG. 9, it was found that the Wild-type (Wild-type) normal mice, the CHM model group disease mice and AAV-treated mice, i.e., the post-treatment mice, respectively, were chM model group CHM ^fl/+ The total retinal thickness of Six3-Cre mice was thinner than that of the wild type group, and the retinal thickness was increased to some extent after AAV treatment, and the structural morphology was also more pronounced recovered than that of the untreated group.

The sequences used in the above examples of the present application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences of embodiments of the present application and are not intended to be limiting in any way. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences, wherein "T" represents uridine when it represents an RNA sequence.

Sequence listing

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Claims

1. An engineered nucleic acid molecule that can express a CHM protein (CHM Rab escort protein) or fragment thereof in a eukaryotic cell, said nucleic acid molecule comprising a coding sequence encoding a sequence as set forth in SEQ ID NO:10, or an amino acid sequence as set forth in SEQ ID NO:10, and a conservative substitution variant of the amino acid sequence shown in seq id no.

2. The engineered nucleic acid molecule of claim 1, the coding sequence comprising the sequence set forth in SEQ ID NO:4, and a polynucleotide sequence shown in seq id no.

3. The engineered nucleic acid molecule of claim 1 or 2, further comprising a 5'utr sequence on the 5' side of the coding sequence.

4. The engineered nucleic acid molecule of claim 3, the 5' utr sequence comprising the sequence set forth in SEQ ID NO:7 and/or SEQ ID NO:8, and a polynucleotide sequence shown in SEQ ID NO.

5. The engineered nucleic acid molecule of claim 3, the 5' utr sequence comprising the sequence set forth in SEQ ID NO: 9.

7. An engineered nucleic acid molecule that can express a CHM protein or fragment thereof in a eukaryotic cell, comprising, in order from 5 'to 3' end, a CMV promoter, a 5'utr, a Kozak sequence, and a CHM coding sequence, wherein the 5' utr sequence comprises the amino acid sequence as set forth in SEQ ID NO:7 and/or SEQ ID NO:8, and a polynucleotide sequence shown in SEQ ID NO.

8. A viral particle comprising the engineered nucleic acid molecule of any one of claims 1-7.

9. The viral particle of claim 8, which is an AAV viral particle.

10. The viral particle according to claim 9, wherein the capsid protein serotype is AAV8.