Engineered cell preparations for treatment of niemann pick b disease

Abstract

The present disclosure provides methods and compositions for culturing, engineering, and administering engineered B lineage cell populations that produce Sphingomyelin Phosphodiesterase 1 (SMPD1).

Description

W O 2 0 2 4 / 2 2 0 4 4 7 A 2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachusetts 02139 (US). FIORINI, Claudia; c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachu- setts 02139 (US). ARLAUCKAS, Sean Philip Leary; c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachusetts 02139 (US). MOR- GAN, Richard A.; c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachu- setts 02139 (US). (43) International Publication Date 24 October 2024 (24.10.2024) (10) International Publication Number WO 2024 / 220447 A2 WIPOIPCT (51) International Patent Classification: Not classified (21) International Application Number: PCT / US2024 / 024830 (22) International Filing Date: 16 April 2024 (16.04.2024) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 63 / 459.983 17 April 2023 (17.04.2023) US 63 / 502,636 16 May 2023 (16.05.2023) US 63 / 592.497 23 October 2023 (23.10.2023) US 63 / 549.943 05 February 2024 (05.02.2024) US 63 / 553,522 14 February 2024 (14.02.2024) US (71) Applicant: BE BIOPHARMA, INC. [US / US]; One Kendall Square, Building 200, Third Floor, Cambridge, Massachusetts 02139 (US). (72) Inventors: MULLEN, Timothy James; c / o Be Biophar- ma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachusetts 02139 (US). DATTA, Ishara; c / o Be Biopharma, Inc., One Kendall Square, Build- ing 200, Third Floor, Cambridge, Massachusetts 02139 (US). LIU, Hanlan; c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachu- setts 02139 (US). TU, Huy Anh; c / o Be Biopharma, Inc., One Kendall Square, Building 200, Third Floor, Cambridge, Massachusetts 02139 (US). HOHMANN, Anja Fides: (74) Agent: ADAMS, Melissa M. et al.; CHOATE, HALL & STEWART LLP, Two International Place, Boston, Massa- chusetts 02110 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW., BY, BZ, CA, CH, CL, CN, CO, CR, CU, CV, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT. HN, HR, HU, ID, IL, IN, IQ, IR, IS, IT, JM, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, MG, MK, MN, MU, MW, MX, MY, MZ, NA, NG, NI, NO. NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO. RS, RU. RW. SA, SC, SD, SE, SG, SK, SL, ST. SV, SY. TH. TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, WS. ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, CV, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SC, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, (54) Title: ENGINEERED CELL PREPARATIONS FOR TREATMENT OF NIEMANN PICK B DISEASE WT SMPD1 BGH polyA g00006 1 endogenous CCR5 locus engineered CCR5 locus FIG. 1A (57) Abstract: The present disclosure provides methods and compositions for culturing, engineering, and administering engineered B lineage cell populations that produce Sphingomyelin Phosphodiesterase 1 (SMPD1). [Continued on next page] 本揭露提供了培養、改造和施用產生鞘磷脂磷酸二酯酶 1 (SMPD1) 的工程化 B 細胞 系群體的方法和組合物。 摘要

Claims

CLAIMS1. A method of preparing an engineered B lineage cell population that expressSphingomyelin Phosphodiesterase 1 (SMPD1), the method comprising the steps of:(a) isolating primary B cells so that a primary B cell population is obtained;(b) activating the primary B cell population; and(c) during or after the activating step, engineering the primary B cell population by integrating a transgene encoding SMPD 1 at a site selected from a CCR5 site, a IgH site, and a JCHAIN site, thereby generating an engineered B lineage cell population.

2. The method of claim 1, further comprising a step of expanding the engineered B lineage cell population.

3. The method of claim 1 or 2, further comprising a step of differentiating the engineered B lineage cell population into a population of plasmablasts.

4. The method of claim 3, wherein the differentiation step comprises contacting the engineered B lineage cell population with culture media comprising:(a) IL-2;(b) IL-6;(c) IL- 10; and / or(d) IL-15.

5. The method of claim 3 or 4, further comprising a step of differentiating the population of plasmablasts into a population of plasma cells.

6. The method of claim 5, wherein the differentiation step comprises contacting the population of plasmablasts with culture media comprising:(a) IL-6;(b) IL- 15; and / or(c) IFN-alpha-2-beta (IFNa-2P).

7. The method of any one of the above claims, further comprising a step of transferring the engineered B lineage cell population to cell culture media without human or bovine serum for about 24 hours.

8. The method of any one of the above claims, wherein the step of engineering further comprises introducing a donor construct comprising the transgene into the primary B cell population.

9. The method of claim 8, wherein the donor construct comprises:(a) a 5’ homology aim that is at least 95% identical to a sequence 5’ to the doublestranded break site; and(b) a 3’ homology aim that is at least 95% identical to a sequence 3’ to the doublestranded break site.

10. The method of claim 9, wherein the 5’ homology arm comprises or is:(i) a length about 450-850 base pairs in size;(ii) a PAM site or absence of a PAM site; and / or(iii) symmetrical or asymmetrical in length with the 3’ homology arm.

11. The method of claim 9 or 10, wherein the 3’ homology arm comprises or is:(i) a length about 450-850 base pairs in size;(ii) a PAM site or absence of PAM site; and / or(iii) symmetrical or asymmetrical in length with the 5 ’ homology arm.

12. The method of claim 9, wherein the 5’ homology aim has at least 95% identity with one or more of SEQ ID NOs: 1-5.

13. The method of claim 9, wherein the 3’ homology aim has at least 95% identity with one or more of SEQ ID NOs: 6-9.

14. The method of claim 8, wherein the donor construct comprises a sequence with at least 95% identity with one or more of SEQ ID NOs: 10-19.

15. The method of claim 14, wherein the construct comprises one or more of:(a) Kozak sequence of 5’-gccacc-3’;(b) a promoter sequence selected from SEQ ID NOs: 20-21;(c) a nucleic acid sequence encoding the SMPD1 selected from SEQ ID NOs: 22-26; and(d) a polyA sequence selected from SEQ ID NOs: 27-28.

16. The method of any one of claims 8-15, wherein the donor construct is or comprises an adeno-associated viral (AAV) vector.

17. The method of any one of the above claims, wherein the step of engineering comprises contacting the primary B cell population with a targeted nuclease capable of introducing doublestranded breaks.

18. The method of claim 17, wherein the targeted nuclease is or comprises a CRISPR- associated (Cas) protein, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALEN), or meganuclease.

19. The method of claim 18, wherein the Cas protein is or comprises Cas9, Casl2a, or Cas 13a, or a variant thereof.

20. The method of claim 18, wherein the Cas protein is complexed with a guide RNA (gRNA).

21. The method of claim 20, wherein the gRNA is a single guide RNA (sgRNA).

22. The method of claim 21, wherein the guide RNA comprises at least 95% identity with any one of SEQ ID NOs: 29-33.

23. The method of any one of the above claims, wherein the step of engineering comprises:(a) electroporation of the primary B cell population, and / or(b) transduction of the primary B cell population with a donor construct.

24. The method of claim 23, wherein the electroporation is performed on a composition comprising:(a) the Cas protein complexed with the gRNA; and(b) the primary B cell population.

25. The method of any one of claims 1-24, wherein the CCR5 site has at least 95% identity with SEQ ID NO: 29.

26. The method of any one of claims 1-24, wherein the IgH site has at least 95% identity with SEQ ID NO: 33.

27. The method of any one of claims 1-24, wherein the JCHAIN site has at least 95% identity with one or more of SEQ ID NOs: 30-32.

28. The method of any one of claims 1-27, wherein the transgene has at least 95% identity with one or more of SEQ ID NOs: 22-26.

29. A population of genetically modified B lineage cells comprising a transgene sequence encoding a SMPD1 protein, wherein the transgene is expressed from an endogenous CCR5 locus.

30. The population of genetically modified B lineage cells of claim 29, wherein the cells express SMPD1 protein and at least partially disrupt expression of endogenous CCR5.31 . A population of genetically modified B lineage cells comprising a transgene sequence encoding a SMPD1 protein, wherein the transgcnc is expressed from an endogenous JCHAIN locus.

32. The population of genetically modified B lineage cells of claim 31, wherein the cells express SMPD1 protein without disrupting expression of endogenous JCHAIN.

33. A population of genetically modified B lineage cells comprising a transgene sequence encoding a SMPD1 protein, wherein the transgene is expressed from an endogenous IgH locus.

34. The population of genetically modified B lineage cells of claim 33, wherein the cells express SMPD1 protein without disrupting expression of endogenous IgH.

35. The population of any one of claims 29-34, wherein the population of genetically modified B lineage cells is or comprises a population of genetically modified plasma cells.

36. The population of any one of claims 29-34, wherein the population of genetically modified B lineage cells is or comprises a population of plasmablasts.

37. The population of claim 36, wherein the population of genetically modified B lineage cells is or comprises a population of plasma cell precursors.

38. A pharmaceutical composition comprising one or more B lineage cells selected from the population of genetically modified B lineage cells of any of the above claims, and one or more pharmaceutically acceptable excipients.

39. A method of administering a pharmaceutical composition, wherein the pharmaceutical composition comprises:(a) one or more B lineage cells selected from the population of genetically modified B lineage cells of any one of the above claims; and(b) one or more pharmaceutically acceptable excipients;wherein the pharmaceutical composition is administered to a subject.

40. A method of treating a disease, disorder, or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of claim 38, thereby treating the disease, disorder, or condition in the subject.

41. The method of claim 39 or 40, wherein the pharmaceutical composition is administered intravenously.

42. The method of any one of claims 39-41, wherein the pharmaceutical composition is administered to an adult subject.

43. The method of any one of claims 39-41, wherein the pharmaceutical composition is administered to a pediatric subject.

44. The method of any one of claims 39-43, wherein the pharmaceutical composition is administered directly to the central nervous system.

45. The method of any one of claims 39-44, wherein the subject has Niemann Pick Disease.

46. A method of characterizing a population of genetically modified B lineage cells of any one of the above claims, comprising assessing one or more of (a)-(g) using an assay:(a) presence of a CD38 marker;(b) presence of a CD 138 marker;(c) presence of at least 50% of a CD27 marker;(d) secretion of at least 0.5 pg / cell / day of IgG;(e) secretion of at least 2.5 pg / cell / day of IgM;(f) secretion of at least 0.5 pg / cell / day of IgD; and(g) secretion of at least 100-300 ng / 106cells / day of a SMPD1 protein.

47. The method of claim 46, wherein the assay comprises or is one or more of: fluorescence- activated cell sorting (FAC-sort), Western Blot, flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), and enzyme-linked immunosorbent assay (ELISA).

48. The method of claim 46 or 47, wherein one or more B lineage cells selected from the population of genetically modified B lineage cells engraft within the bone marrow of the subject.

49. A method of monitoring engraftment of genetically modified B lineage cells within a subject, comprising one or more of: bioluminescence, ELISpot, flow cytometry, and enzyme- linked immunosorbent assay.

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