IP Library › Granted Patent US 12,359,217
Granted Patent B2
US 12,359,217 · App. 17/055,151 · Granted Jul 15, 2025

Virus vector production

Inventors: Alessio Cantore (Milan, IT); Andrea Annoni (Milan, IT); Michela Milani (Milan, IT); Luigi Naldini (Milan, IT)
Assignees: Ospedale San Raffaele S.r.l.; Fondazione Telethon ETS
C12N15/86C12N5/0636C12N7/00C12N2510/02C12N2740/10034C12N2740/10052C12N2740/15034C12N2740/15052C12N2740/16034C12N2740/16043C12N2740/16052
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,359,217
App. No.
17/055,151
Granted
Jul 15, 2025
Kind
B2
Abstract

An enveloped viral particle producer or packaging cell, wherein the cell is genetically engineered to decrease expression of CD47 on the surface of the cell.

Claims (24)

1. An enveloped viral particle producer or packaging cell, wherein:

(a) the producer or packaging cell is genetically engineered to suppress expression of cluster of differentiation 47 (CD47); or

(b) the producer or packaging cell comprises a genetically engineered disruption of a gene encoding CD47,

to decrease expression of CD47 on the surface of the cell, and

wherein the producer or packaging cell comprises one or more nucleic acid sequences encoding one or more viral particle structural proteins.

2. The enveloped viral particle producer or packaging cell of claim 1 , wherein the cell is further genetically engineered to decrease expression of major histocompatibility complex I (MHC-I) on the surface of the cell.

3. The enveloped viral particle producer or packaging cell of claim 1 , wherein the cell comprises a genetically engineered disruption of a gene encoding β2-microglobulin and/or a genetically engineered disruption of one or more genes encoding an MHC-I α chain.

4. The enveloped viral particle producer or packaging cell of claim 1 , wherein the cell is a human embryonic kidney 293 (HEK-293) cell or a derivative thereof.

5. The enveloped viral particle producer or packaging cell of claim 1 , wherein the enveloped viral particle is a retroviral, herpes simplex viral, vaccinia viral, hepadnaviral, togaviral, flaviviral, arenaviral, coronaviral, orthomyxoviral, paramyxoviral, bunyaviral, bornaviral, rhabdoviral or filoviral particle, or a viral particle derived therefrom.

6. The enveloped viral particle producer or packaging cell of claim 1 , wherein the enveloped viral particle is a retroviral, herpes simplex viral or vaccinia viral particle, or a viral particle derived therefrom.

7. A method of producing enveloped viral particles comprising the steps of:

(a) providing an enveloped viral particle producer cell according to claim 1 ; and

(b) culturing the cell under conditions suitable for the production of the enveloped viral particles.

8. An enveloped viral particle obtainable by the method of claim 7 .

9. The enveloped viral particle of claim 8 , wherein the viral particle is a retroviral, herpes simplex viral or vaccinia viral particle, or a viral particle derived therefrom.

10. An isolated cell transduced by the enveloped viral particle of claim 8 .

11. A pharmaceutical composition comprising the enveloped viral particle of claim 8 or comprising an isolated cell transduced by said enveloped viral particle, and a pharmaceutically-acceptable carrier, diluent or excipient.

12. A method of treatment of cancer, bacterial or viral infection, an immune-mediated disease or autoimmune disease comprising transducing an isolated cell with the enveloped viral particle of claim 8 .

13. A method of treatment of cancer, bacterial or viral infection, an immune-mediated disease or autoimmune disease comprising administering the enveloped viral particle of claim 8 , or administering an isolated cell transduced by the enveloped viral particle, to a subject in need thereof.

14. A method of vaccination comprising administering the enveloped viral particle of claim 8 to a subject in need thereof.

15. The enveloped viral particle producer or packaging cell of claim 1 , wherein the cell is a HEK-293T or a HEK-293 T-REx cell.

16. The enveloped viral particle producer or packaging cell of claim 1 , wherein the enveloped viral particle is a lentiviral particle or a viral particle derived therefrom.

17. The enveloped viral particle of claim 8 , wherein the enveloped viral particle is a lentiviral particle or a viral particle derived therefrom.

18. The enveloped viral particle producer or packaging cell of claim 1 , wherein the number of surface-exposed CD47 molecules is less than about 10% of the number of surface-exposed CD47 molecules that are displayed in the absence of the genetic engineering.

Assignments (2)
CHANGE OF NAME Recorded Jun 9, 2025
From: FONDAZIONE TELETHON
To: FONDAZIONE TELETHON ETS
Reel/Frame 071499/0297 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 17, 2024
From: CANTORE, ALESSIO; ANNONI, ANDREA; MILANI, MICHELA; NALDINI, LUIGI
To: OSPEDALE SAN RAFFAELE S.R.L.; FONDAZIONE TELETHON
Reel/Frame 067138/0805 →
Priority Claims (1)
GB 1807945 · May 16, 2018 · national
Continuity (1)
Related Publication 20210222197A1 · Jul 22, 2021
References Cited (78)
US 6329201B1 · Polo · 2001 [cited by examiner]
US 10912824B2 · Cantore · 2021 [cited by examiner]
US 20100316570A1 · Discher et al. · 2010 [cited by applicant]
US 20180214524A1 · Weissman · 2018 [cited by examiner]
US 20180355032A1 · Roberts · 2018 [cited by examiner]
US 20190078096A1 · Lahusen et al. · 2019 [cited by applicant]
US 20210277354A1 · Annoni et al. · 2021 [cited by applicant]
US 20210346489A1 · Cantore · 2021 [cited by examiner]
CN 112601811A · 2021 [cited by applicant]
IN 201817003010 · 2018 [cited by applicant]
WO WO9805635A1 · 1998 [cited by applicant]
WO WO9807859A2 · 1998 [cited by applicant]
WO WO9809985A2 · 1998 [cited by applicant]
WO WO9817815A1 · 1998 [cited by applicant]
WO WO2009131453A1 · 2009 [cited by applicant]
WO WO2014124028A1 · 2014 [cited by applicant]
WO WO2015092440A1 · 2015 [cited by examiner]
WO WO2016009326A1 · 2016 [cited by applicant]
WO WO2017019848A1 · 2017 [cited by applicant]
WO WO2017088012A1 · 2017 [cited by applicant]
WO WO2017180519A1 · 2017 [cited by applicant]
WO WO2017184553A1 · 2017 [cited by applicant]
WO WO2019086574A1 · 2019 [cited by examiner]
Matsunaga, Y. et al. (2008). Activation of Antigen-Specific Cytotoxic T Lymphocytes by β2-Microglobulin or TAP1 Gene Disruption and the Introduction of Recipient-Matched MHC Class I Gene in Allogeneic Embryonic Stem Cel… [cited by examiner]
Gornalusse, G. G. et al. (2017). HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nature Biotechnology, 35(8), 765-772. (Year: 2017). [cited by examiner]
Cantore, A., Milani, M., Lengler, J., Bartolaccini, S., Di Tomaso, T., Gregory, P. D., Scheiflinger, F., Lombardo, A., & Naldini, L. (2015). 6. Targeted Genome Editing of Cell Lines for Improved and Scalable Production … [cited by examiner]
Cantore, A. et al. (2016). 286. Genome Editing of Inducible Cell Lines for Scalable Production of Improved Lentiviral Vectors for Human Gene Therapy. Molecular Therapy, 24, S115-S115. (Year: 2016). [cited by examiner]
Cantore, A., Milani, M., Annoni, A., Liu, T., Bartolaccini, S., Biffi, M., Russo, F., Peters, R., Lombardo, A., Nichols, T. C., Ayuso, E., & Naldini, L. (2017). Liver-Directed Gene Therapy for Hemophilia B with Immune S… [cited by examiner]
Aiuti et al., Gene therapy for immunodeficiency due to adenosine deaminase deficiency, N. Engl. J. Med., 360(5):447-58 (Jan. 2009). [cited by applicant]
Aiuti et al., Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome, Science, 341(6148):1233151 (Aug. 2013). [cited by applicant]
Amabile et al., Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing, Cell, 167(1):219-232.e14 (Sep. 2016). [cited by applicant]
Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, chapters 9, 13, 16 (1995). [cited by applicant]
Benechet et al., Intravital Microscopy Analysis of Hepatic T Cell Dynamics, Methods Mol. Biol., 14:49-61 (2017). [cited by applicant]
Biffi et al., Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy, Science, 341(6148):1233158 (Aug. 2013). [cited by applicant]
Bobis-Wozowicz et al., Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery, Scientific Reports, 4, article No. 4656 (2014). [cited by applicant]
Boztug et al., Stem-cell gene therapy for the Wiskott-Aldrich syndrome, N. Engl. J. Med., 363(20):1918-27 (Nov. 2010). [cited by applicant]
Cantore et al., Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice, Blood, 120(23):4517-20 (2012). [cited by applicant]
Cantore et al., Liver-directed lentiviral gene therapy in a dog model of hemophilia B, Science Translational Medicine, 7, pp. 277ra28 (2015). [cited by applicant]
Cartier et al., Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy, Science, 326(5954):818-23 (Nov. 2009). [cited by applicant]
Coffin et al., Retroviral taxonomy, protein structures, sequences and genetic maps, IN: Retroviruses, Cold Spring Harbour Laboratory Press, pp. 758-763 (1997). [cited by applicant]
Ewer et al. , A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA, New England J Medicine, 2015, vol. 374, pp. 1635-1646. [cited by applicant]
Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press (1984). [Table of Contes]. [cited by applicant]
Gaj et al, ZFN, TALEN and CRISPR/Cas-based methods for genome engineering, Trends Biotechnol., vol. 31, pp. 397-405 (2013). [cited by applicant]
GenBank Accession No. NM_004048, May 3, 2014. [cited by applicant]
Hacien-Bey-Abina et al., Efficacy of gene therapy for X-linked severe combined immunodeficiency, N. Engl. J. Med., 363(4):355-64 (Jul. 2010). [cited by applicant]
International Application No. PCT/EP2019/062664, International Search Report and Written Opinion, mailed Aug. 19, 2019. [cited by applicant]
Koh et al., Exosome-SIRPa, a CD47 blockade increases cancer cell phagocytosis, Biomaterials, 2017, No. 121, pp. 121-129. [cited by applicant]
Leavitt et al., Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection, J Virol, vol. 70, pp. 721-728 (1996). [cited by applicant]
Lee et al., CD47 Plays a role as a negative regulator in inducing protective immune responses to vaccination against influenza virus, Journal of Virology, Aug. 2016, vol. 90, No. 15, pp. 6746-6758. [cited by applicant]
Lewis et al., Human immunodeficiency virus infection of cells arrested in the cell cycle, EMBO J, vol. 11, pp. 3053-3058 (1992). [cited by applicant]
Lewis et al., Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus, J Virol, vol. 68, pp. 510-516 (1994). [cited by applicant]
Lichty et al., Going viral with cancer immunotherapy, Nat. Rev. Cancer, 14(8):559-67 (2014). [cited by applicant]
Lilley et al. (eds.), DNA Structures, Part A, Synthesis and Physical Analysis of DNA, vol. 211 in Methods in Enzymology, San Diego, California: Academic Press, Inc. (1992). [cited by applicant]
Lombardo et al., Site-specific integration and tailoring of cassette design for sustainable gene transfer, Nat Methods, 2011, vol. 8, pp. 861-869. [cited by applicant]
Maetzig et al., Retroviral protein transfer: falling apart to make an impact, Current Gene Ther, vol. 12, pp. 389-409 (2012). [cited by applicant]
Matsui et al., A microRNA-regulated and GP64-pseudotyped lentiviral vector mediates stable expression of FVIII in a murine model of Hemophilia A, Mol. Ther., 19(4):723-30 (Apr. 2011). [cited by applicant]
Milani et al., Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy, EMBO Mol. Med., 9(11):1558-73 (Nov. 2017). [cited by applicant]
Milani et al., Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates, Sci. Transl. Med, May 22, 2019, vol. 11(493): eaav7325. [cited by applicant]
Naldini et al., Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector, Proc. Natl. Acad. Sci. USA, vol. 93, pp. 11382-11388 (1996). [cited by applicant]
Naldini et al., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science, vol. 272, pp. 263-267 (1996). [cited by applicant]
Nightingale et al., Transient gene expression by nonintegrating lentiviral vectors, Mol. Ther, vol. 13, pp. 1121-1132 (2006). [cited by applicant]
Oldenborg et al., Role of CD47 as a Marker of Self on Red Blood Cells, Science, Jun. 16, 2000, vol. 288(5473), pp. 2051-2054. [cited by applicant]
Penn, Major Histocompatibility Complex (MHC), Encyclopedia of Life Sciences, John Wiley & Sons (2005). [cited by applicant]
Polak et al. (eds.), In Situ Hybridization: Principles and Practice, New York: Oxford University Press (1990) [Table of Contents]. [cited by applicant]
Rodriguez et al., Minimal “Self” Peptides That Inhibit Phagocytic Clearance and Enhance Delivery of Nanoparticles, Science, Feb. 22, 2013, vol. 339, pp. 971-974. [cited by applicant]
Roe et al., DNA Isolation and Sequencing: Essential Techniques, Chichester, West Sussex: John Wiley & Sons (1996). [Table of Contents]. [cited by applicant]
Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Harbor Laboratory, 2nd edition (1989), [Table of Contents]. [cited by applicant]
Sosale et al., “Marker of Self” CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors, Molecular Therapy—Methods & Clinical Development, 3:16… [cited by applicant]
Sosale et al., Reducing Immune Response against Lentiviral Vectors; Lentiviral Vector Presentation of CD47, the “Marker of Self”, Biophysical Journal, 2011, vol. 100, issue 3, supplement 1, p. 403a, abstract 2181-Pos Bo… [cited by applicant]
Sosale, Inhibiting Phagocytosis with Cd47: from the effects of red cell rigidity and shape to display on lentivirus—implications for aging and gene therapy, University of Pennsylvania Dissertations, Jan. 1, 2014, No. 14… [cited by applicant]
Tseng et al., Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response, Proc Natl Acad Sci., Jul. 2, 2013, vol. 110, No. 27, pp. 11103-11108. [cited by applicant]
Voelkel et al., Protein transduction from retroviral Gag precursors, Proc National Acad Sci USA, vol. 107, No. 17, pp. 7805-7810 (2010). [cited by applicant]
Wu et al., Critical role of integrin CD11c in splenic dendritic cell capture of missing-self CD47 cells to induce adaptive immunity, Proc. Natl. Acad. Sci. USA, 115(26):6786-91 (Jun. 2018). [cited by applicant]
Cantore et al., Efficacy and safety of liver-directed lentiviral gene therapy in hemophilia B dogs and non-human primates, Mol. Ther., 25(5 Suppl 1):30-31 Abstract (2017). [cited by applicant]
Milone et al., Clinical use of lentiviral vectors, Leukemia, 32(7):1529-41 (2018). [cited by applicant]
Toledano et al., Novel CD47: SIRPα dependent mechanism for the activation of STAT3 in antigen-presenting cell, PLoS One, 8(9):e75595 (2013). [cited by applicant]
Bai et al., Achievements and concerns of the CD47 targeted anti-cancer therapy, Chin J Clin Oncol., 44(7): 344-348 (2017). [cited by applicant]
Gao et al., Effect of small interfering RNA targeting CD47 gene mediated by lentivirus vectors on proliferation and apoptosis of human laryngocarcinoma Hep-2 cells, Med J Chin PLA, 38(8): 634-638 (2013). [cited by applicant]