CN-122012625-A - Viral vector production

Abstract

The present invention relates to viral vector production. In particular, the present invention provides novel methods for generating viral vectors. Corresponding viral vector production systems and uses are also provided.

Inventors

• Rui Andre Sareva Raposo
• Charles Moore Kelly
• Jordan White
• DANIEL FARLEY

Assignees

• 牛津生物医学（英国）有限公司

Dates

Publication Date

20260512

Application Date

20210514

Priority Date

20200515

Claims (10)

1. 1. A method for producing a viral vector, the method comprising culturing cells comprising a nucleic acid sequence encoding a viral vector component in a cell culture medium comprising a PKC activator.
2. 2. The method of claim 1, wherein the viral vector is a self-inactivating viral vector.
3. 3. The method of any one of the above claims, wherein the PKC activator is prostratin or phorbol 12-myristate 13-acetate, an analog, derivative, or pharmaceutically acceptable salt thereof.
4. 4. A method according to claim 3, wherein: a) The prostratin is in the cell culture medium at a concentration of at least about 0.5. Mu.M, optionally wherein the prostratin is at a concentration of about 0.5 to about 32. Mu.M, or B) Phorbol 12-myristate 13-acetate is in the cell culture medium at a concentration of at least about 1nM, optionally wherein phorbol 12-myristate 13-acetate is at a concentration of about 1 to about 32 nM.
5. 5. The method of any one of the above claims, wherein the viral vector is a lentiviral vector and the modified U1 snRNA is co-expressed with the lentiviral vector component, wherein the modified U1 snRNA binds to a nucleotide sequence within a packaging region of the lentiviral vector genomic sequence.
6. 6. The method of any one of the above claims, wherein the viral vector is a lentiviral vector and wherein splice activity from a major splice donor region of the lentiviral vector genome has been functionally eliminated.
7. 7. The method of any one of the above claims, wherein the viral vector is a lentiviral vector, wherein the lentiviral vector genome has been mutated in the major splice donor region or mutated in the major splice donor region and at least one cryptic splice donor region.
8. 8. The method of any one of the above claims, wherein the cell culture medium further comprises an HDAC inhibitor.
9. 9. The method of claim 8, wherein the HDAC inhibitor is an aliphatic HDAC inhibitor or a hydroxamic acid HDAC inhibitor.
10. 10. The method of claim 9, wherein the aliphatic HDAC inhibitor is sodium butyrate, sodium valproate, or valeric acid, an analog, derivative, or pharmaceutically acceptable salt thereof.

Description

Viral vector production The present application is a divisional application of Chinese patent application No. 202180035199.X entitled "production of viral vectors" with application day 2021, month 05 and 14. Technical Field The present invention provides novel methods for producing viral vectors. Corresponding viral vector production systems and uses are also provided. Background The development and production of viral vectors for vaccines and human gene therapies has been well documented in the past few decades, in the scientific journal and patents. The use of engineered viruses for therapeutic delivery of transgenes is broad. Modern Gene therapy vectors based on RNA Viruses, such as gamma-retrovirus and lentivirus (Muhlebach, m.d. et al , 2010, Retroviruses: Molecular Biology, Genomics and Pathogenesis, 13:347-370;Antoniou, M.N., Skipper, K.A. & Anakok, O., 2013, Hum. Gene Ther.,24:363-374） and DNA Viruses, such as adenovirus (Capasso, c. et al 2014, viruses, 6:832-855) and adeno-associated virus (AAV) (Kotterman, m.a. & schafer, d.v., 2014, nat. Rev. Gene, 15:445-451) have shown promise in an increasing number of human disease indications, these include the use of patient cells for the production of large scale human disease conditions (Morgan, r.a. & Kakarla, s., caner j., 20:145-150; touzot, f. Et al 2014, expert opin. Biol. Ter., 14:789-798), and ophthalmology (Balaggan, k.s. & Ali, r.r., the Gene., 19:145-153), cardiovascular (Katz, m.g. 3, r.p. 2012), 35, and 35.d. 25-35, and the use of such vectors for the clinical treatment of large scale retrogression of human diseases (35, r.35, r.v. 35-35), and the use of such vectors has been successfully approved in the production of large scale human disease conditions (e.35, such as well-established by the use of patient cells in the clinical laboratory scale of vascular conditions (Morgan, r.a.) and the production of the vectors of the drugs, such as well as the large-scale human diseases). The way to overcome this difficulty is to find new methods to maximize titers during production of viral vectors. Conventional methods of viral vector production include transfection of primary cells or mammalian/insect cell lines with vector DNA components followed by a limited incubation period, followed by harvesting of the crude vector （Merten, O-W., Schweizer, M., Chahal, P., & Kamen, A.A., 2014, Pharmaceutical Bioprocessing, 2:183-203）. from the medium and/or cells otherwise, the use of producer cell lines during a transfection-independent method (PrCL; wherein all necessary vector component expression cassettes are stably integrated into producer cell DNA), which is advantageous on a larger scale. The efficiency of viral vector production at the "upstream stage" is generally affected by several factors including [1] the serotype/pseudotype of virus used, [2] the transgene sequence composition and size, [3] the medium composition/aeration/pH, [4] the transfection reagent/method, [5] the chemical induction and timing of vector harvest, [6] cell vulnerability/viability, [7] bioreactor shear and [8] impurities. Clearly, there are still other factors to consider in the "downstream" purification/concentration stage (Merten, O-W. Et al 2014, pharmaceutical Bioprocessing, 2:237-251). Accordingly, there is a need in the art to provide alternative methods of producing viral vectors that help solve the problems known to be associated with the large-scale production of GMP-grade vector materials. Disclosure of Invention The inventors surprisingly showed that the use of PKC activators alone or in combination with HDAC inhibitors significantly increased viral vector titres during viral vector production. Thus, the present invention relates to the use of PKC activators i) as inducers of viral vector production by themselves and ii) as enhancers of HDAC inhibitor induction of viral vector production. The inventors have also shown that cells treated with PKC activators maintain high cell viability, which is beneficial during viral vector production. Thus, there is provided a method for producing a viral vector, the method comprising culturing cells comprising a nucleic acid sequence encoding a viral vector component in a cell culture medium comprising a PKC activator. Suitably, the viral vector may be a self-inactivating viral vector. Suitably, the PKC activator may be prostratin or phorbol 12-myristate 13-acetate, an analogue, derivative or pharmaceutically acceptable salt thereof. Suitably the first and second support members are arranged, A) The prostratin may be in the cell culture medium at a concentration of at least about 0.5. Mu.M, alternatively wherein the prostratin may be at a concentration of about 0.5 to about 32. Mu.M, or B) Phorbol 12-myristate 13-acetate may be in the cell culture medium at a concentration of at least about 1nM, optionally wherein phorbol 12-myristate 13-acetate may be at a concentration of about 1 to about 32 nM. Suitably, the viral vector may b