Point-of-care and/or portable platform for gene therapy
A platform for ex vivo isolation, production, and formulation of genetically-modified cells is described. The platform utilizes a software-enabled point-of-care and/or portable device making gene therapy more widely available.
1. A method performed by a device, the method comprising:
receiving, by a treatment chamber, a subject sample comprising at least one first cell type and at least one second cell type;
transferring the subject sample from the treatment chamber to at least one cell type separator;
separating the at least one first cell type in the subject sample by allowing the at least one second cell type to pass through the cell type separator and retaining the at least one first cell type within the at least one cell type separator;
transferring the at least one first cell type into the treatment chamber;
introducing a genetic modifier to the at least one first cell type to generate genetically-modified cells;
pelleting the genetically-modified cells within the treatment chamber through centrifugation to create a pelleted cell suspension;
removing a specified supernatant volume from the pelleted cell suspension through at least one first conduit connected to the treatment chamber;
diluting the pelleted cell suspension with media comprising a pharmaceutically acceptable carrier;
centrifuging the diluted pelleted cell suspension and removing an additional volume of supernatant in the treatment chamber through the at least one first conduit to form a final cell product formulation; and
transferring the final cell product formulation into one or more sterile receptacles through a second conduit connected to the treatment chamber wherein the final cell product formulation is ready for administration to a subject upon completion of transfer into the one or more sterile receptacles.
2. The method of claim 1 , wherein a user of the device:
determines an initial volume of the subject sample;
determines a hematocrit level of the subject sample;
determines a volume of buffer to add to the subject sample to reduce the hematocrit level to at least 25% according to the following formula:
(
starting
product
volume
(
mL
)
×
obtained
hematocrit
value
(
%
)
25
%
desired
hematocrit
value
)
-
starting
product
volume
(
mL
)
;
and wherein the device adds the determined volume of buffer to the subject sample.
3. The method of claim 1 , comprising performing release testing to verify compliance with Current Good Manufacturing Practices wherein the release testing comprises
Test
Required Result
Gram Stain
Negative
3 Day Sterility
Negative
14 Day Sterility
Negative
Mycoplasma
Negative
Endotoxin
≤0.5 EU/ml
Cell Viability by Trypan Blue Dye Exclusion
≥70%.
4. The method of claim 1 , wherein the introducing of the genetic modifier inserts or alters a gene selected from ABCD1, ABCA3, ABLI, ADA, AKT1, APC, APP, ARSA, ARSB, BCL11A, BLC1, BLC6, BRCA1, BRCA2, BRIP1, C9ORF72, C46, CAR, CAS9, C-CAM, CBFAI, CBL, CCR5, CD4, CD19, CD40, CDA, CFTR, CLN3, C-MYC, CRE, CSCR4, CSFIR, CTLA, CTS-I, CYB5R3, DCC, DHFR, DKC1, DLL1, DMD, EGFR, ERBA, ERBB, EBRB2, ETSI, ETS2, ETV6, F8, F9, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FasL, FCC, FGR, FOX, FUS, FUSI, FYN, GALNS, GATA1, GLB1, GNS, GUSB, HBB, HBD, HBE1, HBG1, HBG2, HCR, HGSNAT, HOXB4, HRAS, HYAL1, ICAM-1, iCaspase, IDUA, IDS, JUN, KLF4, KRAS, LCK, LRRK2, LYN, MCC, MDM2, MGMT, MLL, MMACI, MYB, MEN-I, MEN-II, MYC, NAGLU, NANOG, NF-1, NF-2, NKX2.1, NOTCH, OCT4, p16, p21, p27, p53, p57, p73, PALB2, PARK2, PARK7, phox, PINK1, PK, PSEN1, PSEN2, PTPN22, RAD51C, ras, at least one of RPL3 through RPL40, RPLP0, RPLP1, RPLP2, at least one of RPS2 through RPS30, RPSA, SFTPB, SFTPC, SGSH, SLX4, SNCA, SOD1, SOX2, TERC, TERT, TDP43, TINF2, TK, ubiquilin 2, VHL, WAS and WT-1.
5. The method of claim 1 , wherein the genetically-modified cells are hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), hematopoietic stem and progenitor cells (HSPC), T cells, natural killer cells, B cells, macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells, and/or bone marrow fibroblasts.
6. The method of claim 1 , wherein the genetic modifier comprises a lentiviral vector comprising a pseudotype envelope glycoprotein and a lentiviral RNA molecule wherein the pseudotype envelope glycoprotein comprises vesicular stomatitis virus glycoprotein (VSVG), cocal virus glycoprotein (cocal), feline endogenous virus glycoprotein (RD114), or modified foamy virus glycoprotein (mFoamy) and wherein the lentiviral RNA molecule comprises a HIV-1-derived, self-inactivating lentivirus backbone which is integration deficient.
7. The method of claim 6 , wherein the genetic modifier comprises a foamy viral vector comprising a pseudotype envelope glycoprotein and a foamy viral RNA molecule wherein the pseudotype envelope glycoprotein comprises foamy viral envelope protein (Foamy), or modified foamy viral envelope protein (mFoamy) and wherein the foamy viral RNA molecule comprises a self-inactivating foamy virus backbone which is integration deficient.
8. The method of claim 1 , wherein the one or more sterile receptacles are sterile-welded to the second conduit.
9. The method of claim 1 , comprising the treatment chamber receiving the subject sample in a number of stages wherein the number of stages is determined by a user of the device and is obtained by diluting the subject sample and dividing the diluted sample volume in mL by 300 mL per stage and rounding up to the next whole number.
10. The method of claim 1 , comprising the device receiving a signal that sedimentation is complete and red blood cell (RBC) removal may begin.
11. The method of claim 1 , comprising the device receiving a signal determining an initial volume in mL to remove from a RBC sediment layer.
12. The method of claim 1 , comprising receiving, by the at least one cell type separator selector beads or antibodies that selectively bind CD3, CD4, CD8, CD13, CD14, CD15, CD16, CD19, CD20, CD34, CD45, CD45RA, CD45RO, CD49f, CD50, CD56, CD71, CD90, or CD133.
13. The method of claim 1 , comprising receiving, by the at least one cell type separator microbeads and intravenous immunoglobulin (IVIg).
14. The method of claim 1 , comprising receiving, by the treatment chamber, transduction media wherein the transduction media comprises
(i) a base medium;
(ii) cyto- and/or chemokines comprising recombinant human granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), flightless 3 ligand (flt3 or flt3L), and/or interleukins selected from interleukin 3 (IL-3) and/or interleukin 6 (IL-6); and
(iii) agents to promote cell survival and gene transfer comprising
(a) aryl-hydrocarbon receptor antagonists selected from StemRegenin1; GNF351; and/or CH223191,
(b) pyrimidoindole derivatives selected from UM171, and/or UM118428,
(c) glucocorticoid receptor antagonists selected from mifepristone, RU-43044, Miconazole, 11-oxa cortisol, 11-oxa prednisolone, and/or dexamethasone mesylate;
(d) protamine sulfate,
(e) rapamycin,
(f) polybrene,
(g) fibronectin fragment,
(h) prostaglandin E2,
(i) antioxidants and/or
(j) nonsteroidal anti-inflammatory drugs selected from celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, and/or tolmetin.
15. The method of claim 1 , further comprising tagging, within the treatment chamber and/or the at least one cell type separator the at least one first cell type with immunomagnetic beads,
wherein separating the at least one first cell type comprises retaining the immunomagnetic beads on a magnetically susceptible material,
wherein the magnetically susceptible material is disposed between a first electrode and a second electrode, and
wherein introducing a genetic modifier to the at least one first cell type comprises inducing pores in the at least one first cell type by activating an electric potential between the first electrode and the second electrode when the at least one first cell type are located in a chamber of the cell type separator.
16. A method performed by a device, the method comprising:
receiving, by at least one chamber, a subject sample comprising cells;
introducing a genetic modifier to the cells to generate genetically-modified cells;
pelleting the genetically-modified cells within the at least one chamber through centrifugation to create a pelleted cell suspension;
removing, from the at least one chamber, a supernatant volume from the pelleted cell suspension through at least one conduit connected to the at least one chamber;
diluting the pelleted cell suspension with a media comprising a pharmaceutically acceptable carrier;
centrifuging the diluted pelleted cell suspension and removing an additional volume of supernatant through at least one conduit connected to the at least one chamber to form a final cell product formulation; and
transferring the final cell product formulation into one or more sterile receptacles through at least one conduit connected to the at least one chamber wherein the final cell product formulation is ready for administration to a subject upon completion of transfer into the one or more sterile receptacles.
17. The method of claim 16 , wherein the genetic modifier comprises a lentiviral vector comprising a pseudotype envelope glycoprotein and a lentiviral RNA molecule, wherein the pseudotype envelope glycoprotein comprises VSVG, cocal, RD114, or mFoamy, and wherein the lentiviral RNA molecule comprises a HIV-1-derived, self-inactivating lentivirus backbone which is integration deficient.
18. The method of claim 16 , wherein the genetic modifier comprises a foamy viral vector comprising a pseudotype envelope glycoprotein and a foamy viral RNA molecule wherein the pseudotype envelope glycoprotein comprises Foamy, or mFoamy and wherein the foamy viral RNA molecule comprises a self-inactivating foamy virus backbone which is integration deficient.
19. The method of claim 16 , wherein the genetically-modified cells are HSC, HPC, HSPC, T cells, natural killer cells, B cells, macrophages, monocytes, MSC, WBC, MNC, EC, stromal cells, and/or bone marrow fibroblasts.
20. The method of claim 16 , wherein the genetically-modified cells are CD34+ HSPC.