Adenoviral expression vectors and methods of production and cell lines

By introducing mutants and molecular cloning fragments into adenovirus vectors and combining them with engineered cell lines, the problems of inconvenient adenovirus vector operation and infectious replication were solved, achieving safe and efficient adenovirus vector production and expression.

CN122104807APending Publication Date: 2026-05-29LUNG BIOTECH PBC
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
LUNG BIOTECH PBC
Filing Date
2021-04-06
Publication Date
2026-05-29

AI Technical Summary

Technical Problem

Existing adenovirus vectors are inconvenient to handle in vitro, are prone to uncontrolled replication, and common mammalian cell lines may recombine with mutant vectors to produce infectious replicating viruses. There is a lack of safe, non-infectious, non-replicating vectors and effective expression cassettes, making it difficult to produce them efficiently in bacteria.

Method used

Designing non-replicable adenovirus vectors involves introducing mutations such as E1 or E3 gene deletions, molecularly cloning multiple small fragments in bacteria, expressing E1A and E1B gene products using engineered mammalian cell lines, inhibiting recombination, and combining lentiviral vector selective markers to ensure safe production.

Benefits of technology

This technology enables the efficient production of non-replicating adenovirus vectors in bacteria, avoiding infectious contamination and providing a safe expression system suitable for gene therapy.

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Abstract

Adenovirus expression vectors and production methods and cell lines are provided. The adenovirus expression vectors can include: a) one or more mutations that render the adenovirus replication-incompetent; b) at least one nucleotide sequence encoding a protein or RNA. A method of synthesizing an adenovirus vector is also provided. The synthesis method can include: a) generating a plurality of overlapping adenovirus subfragments, each subfragment comprising a portion of the full genome of the adenovirus; b) circularizing the subfragments to form a plasmid structure; and c) assembling the circularized subfragments into a linear structure, wherein the vector comprises a combination of two or more subfragments. A mammalian cell line configured to replicate an adenovirus vector is also provided, wherein the cell line includes nucleotide sequences expressing E1A and E1B gene products, but does not contain other adenovirus sequences.
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Claims

1. A non-replicating adenovirus expression vector, comprising: a) one or more mutations that prevent adenovirus from replicating, and b) at least one nucleotide sequence encoding a gene product.

2. The vector according to claim 1, wherein the vector comprises a nucleotide sequence having at least 80% sequence identity over the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary sequence thereof.

3. The vector according to claim 2, wherein the vector comprises a nucleic acid sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than 99% sequence identity with SEQ ID NO: 1-2.

4. The vector according to claim 1, wherein the vector comprises an adenovirus serotype 5 vector.

5. The vector according to claim 1, wherein the one or more mutations include the deletion of the E1 gene or the E3 gene or both.

6. The vector according to claim 1, wherein the at least one nucleotide sequence comprises a transgene.

7. The vector according to claim 6, wherein the transgene expresses human IL10.

8. The vector of claim 6, wherein the transgene further comprises at least one of an enhancer / promoter region containing a CAG region, human IL10 cDNA, or a polyadenylation signal, or any combination thereof.

9. The carrier according to claim 8, wherein the polyadenylation signal comprises the SV40 region and / or the complete polyA signal.

10. The vector according to claim 8, wherein the polyadenylation signal is first inserted between nucleotides 440 and 3515 of SEQ ID NO:

3.

11. The vector according to claim 8, wherein the insertion of the transgene replaces the E1 gene but leaves the pIX gene intact.

12. A method for synthesizing an adenovirus vector, comprising: a) Generate multiple overlapping adenovirus sub-fragments, each of which includes a portion of the complete adenovirus genome; b) Circularize the sub-fragments to form a plasmid structure; and c) Assemble the circularized sub-fragments into a linear structure, wherein the carrier comprises a combination of two or more sub-fragments.

13. The method of claim 12, wherein the adenovirus vector comprises adenovirus serotype 5.

14. The method of claim 13, wherein the vector replicates efficiently in bacteria.

15. The method of claim 12, wherein the first sub-fragment comprises the sequence described from the 3' end of SEQ ID NO: 4 or from the 5' end of SEQ ID NO:

5.

16. The method of claim 12, wherein the second sub-fragment comprises the sequence from the 3' end of SEQ ID NO: 6 or the 5' end of SEQ ID NO:

7.

17. The method of claim 12, wherein the right end of the second sub-segment is encoded by SEQ ID NO:

11.

18. The method of claim 12, wherein the left end of the second sub-segment is encoded by SEQ ID NO:

12.

19. The method of claim 12, wherein the 3' end of the second sub-segment is encoded by SEQ ID NO:

13.

20. The method of claim 12, wherein each sub-fragment comprises about 50% of the adenovirus whole genome.

21. The method of claim 20, wherein at least one fragment comprises a transgene expressing human IL-10.

22. The method of claim 21, wherein the transgene further comprises at least one of an enhancer or promoter region, a human IL10 cDNA gene, or a polyadenylation signal, or any combination thereof.

23. The method of claim 22, wherein the enhancer / promoter region comprises a CAG promoter.

24. The method of claim 22, wherein the transgene includes at least a polyadenylation signal comprising an SV40 promoter or a complete poly(A) signal.

25. The method of claim 12, wherein the vector sequence comprises a linear cloning vector backbone, which is removed during the final synthesis of the adenovirus vector.

26. The method of claim 22, wherein the human IL10 cDNA sequence is optimized and encoded by SEQ ID NO:

8.

27. A mammalian cell line configured to replicate an adenovirus vector, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products, but does not contain other adenovirus sequences.

28. The cell line of claim 24, further comprising a lentiviral vector engineered to express E1A and E1B gene products, wherein when the mutated adenovirus vector is introduced into the cell line, the lentiviral vector inhibits the remodeling of the functional adenovirus.

29. The cell line of claim 25, wherein the cell line comprises HeLa cells.

30. The cell line of claim 28, wherein the cell line is generated by transfecting HEK293 cells with a lentiviral genome plasmid comprising at least E1A and E1B coding sequences.

31. The cell line of claim 27, wherein the plasmid containing at least an E1A coding sequence and an E1B coding sequence contains E1A and E1B sequences arranged in a bidirectional expression unit having a bidirectional promoter to prevent recombination with a mutated adenovirus vector.

32. The cell line of claim 28, wherein the bidirectional promoter is a combination of truncated CMV and PGK promoters.

33. The cell line of claim 27, further comprising a puromycin resistance gene in a lentiviral genome sequence, so as to distinguish cells integrated with the lentiviral sequence when expressing the puromycin resistance gene.

34. The cell line of claim 27, wherein the final vector encoded by SEQ ID NO: 9 is used to produce lentivirus, transduce HeLa cells, and select for puromycin resistance.

35. A method for producing the carrier as claimed in claim 1, the method comprising: Transforming mammalian cells with an adenovirus vector comprising a) one or more mutations that prevent adenovirus from replicating, and b) at least one nucleotide sequence encoding a transgene, wherein the mammalian cells comprise nucleotide sequences expressing E1A and E1B gene products but without other adenovirus sequences; culturing the transformed mammalian cells in cell lines containing inserted lentiviral E1A and E1B vectors; and isolating the adenovirus vector.

36. The method of claim 35, wherein the transgene expresses human IL-10.

37. A method of treating a subject who requires a gene product, the method comprising administering a vector to the subject, the vector comprising (a) one or more mutations that prevent adenovirus from replicating and (b) at least one nucleotide sequence encoding the gene product.

38. The method of claim 37, wherein the vector comprises a nucleotide sequence having at least 80% sequence identity over the entire sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a complementary sequence thereof.

39. The method of claim 37, wherein the vector comprises an adenovirus serotype 5 vector.

40. The method of claim 37, wherein the one or more mutations comprise the deletion of the E1 gene or the E3 gene or both.

41. The method of claim 37, wherein the at least one nucleotide sequence comprises a transgene.

42. The method of claim 42, wherein the transgene expresses human IL10.

43. The method of claim 42, wherein the subject is a human.

44. The method of claim 42, wherein the gene product is a peptide.

45. The method of claim 44, wherein the peptide is a human peptide.

46. ​​The method of claim 42, wherein the gene product is RNA.