CA2086844A1 - Method and product of gene transfer into cells - Google Patents
Method and product of gene transfer into cellsInfo
- Publication number
- CA2086844A1 CA2086844A1 CA 2086844 CA2086844A CA2086844A1 CA 2086844 A1 CA2086844 A1 CA 2086844A1 CA 2086844 CA2086844 CA 2086844 CA 2086844 A CA2086844 A CA 2086844A CA 2086844 A1 CA2086844 A1 CA 2086844A1
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- CA
- Canada
- Prior art keywords
- cells
- stem cells
- gene
- cell
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
Abstract
ABSTRACT
A method of effecting transfer of a gene into mammalian cells, particularly, hematopoietic cells with a gene transfer vehicle, particularly a retroviral vector. The method comprises establishing a long term cell culture and exposing the culture to multiple, periodic infections of the vector containing the gene and, preferably, comprising multiple, periodic partial substitutions of the medium and cells. Genetically marked cells are returned to autologous recipients in the absence of any type of conditioning. The method provides improved gene transfer efficiency without increased toxicity.
A method of effecting transfer of a gene into mammalian cells, particularly, hematopoietic cells with a gene transfer vehicle, particularly a retroviral vector. The method comprises establishing a long term cell culture and exposing the culture to multiple, periodic infections of the vector containing the gene and, preferably, comprising multiple, periodic partial substitutions of the medium and cells. Genetically marked cells are returned to autologous recipients in the absence of any type of conditioning. The method provides improved gene transfer efficiency without increased toxicity.
Description
~.$~
~T~OD A~D P~OD~C~ O~ $~N~ T~U~8~R I~O ~
FI~D O~ T~B ~ O~ -Thi~ invention relates to a mçthod of tran~ferring genes into mammalian cellR, particularly, he~atopoietic stem cells, with a gene transfer vector, partic~larly, a retrovirus vector; to resultant mammalian cells produced thereby; method of activatin~ stem cells; method of adoptive transfer of said cells; medicament compositions comprising said ells, methods of ~edical tre~tment u~ing said medicament~, and method~ of gen~ transfer in living organisms for non-medical purposes.
B~GRO~D TO ~Xg I~Vg~IO~
Gene tran~fer technology provide~ a powerful approach to the modification and study of cellular function. Efficient gen.e transfer into the genetic material of living organis~ i5 beao~ing increasingly desira*le and gaining importance. Transfer into plant c~lls, either for dissase control or to add or i~prove a desirable characteristic, is well establi~hed. In the medical field specific applications include the treatment ~ :
of genetic disease,~ cancer therapy, 1~5~6 and characterization of the developmental program of stem 2 ~
cells.7 A list of conditions that ara candidates for gene therapy comprises thalassemias, ADA de~iciency, PNP
deficiency, CGD, lysosomal storage! diseases, leukemias and other cancers~ myelodysplastic syndromes, viral infections, vitamin deficiencies, inborn errors of metabolism, infections and suppression or enhancement of normal functions post therapies for other illne~.
The target in most o these applications is the pluripotent hematopoietic stem cell, but the low frequency and quie~cent nature o~ stem cell~ neces~itates a highly ef~icient gene transfer syst2m. To date, retroviruses have been the most useful vector~ for gene transfer.l~4~8 Foreign genes hav~ been introduced in YitrO
into hematopoietic progenitors of many specie~ including the mouse, ~15 dQg,1~21 nonhuman primate~ ~ and hu~ans.~S~
28 Gene transfer and expression into pluripotent stem cells capable of long-ter~ reconstitution has only been shown ~or the mouse, using a variety of vectors.~ S
Despite these sucsesses, major issues remain to be resolved before therapeutic measures using gene transfer into human hematopoietic cell~ can be considered feasible.
One of these issues is the need to develop methods to transfer genes of interest at high ef~iciency into stem cells capable o~ reconstituting the hematopoietic system o~ large animals. In general, marrow cells infected in vitro have noS been maintained at high levels in vivo after autologous transplantation. Thi~ result has been attributed to the inability to infect a sufficiently large n~mber o~ primitive stem cells with reconstituting capacity.t~26 Mo~t infection protocols involve short-t~erm (1 to 3 day~) cocultivation of BM
cells with virus-containing media, or cocultivation over irradiated monolayers of virus-producing fibroblasts (reviewe~ by Kohn et al4~. Evidenca indicates that cell .. . . . .
- 2~8~4~
~T~OD A~D P~OD~C~ O~ $~N~ T~U~8~R I~O ~
FI~D O~ T~B ~ O~ -Thi~ invention relates to a mçthod of tran~ferring genes into mammalian cellR, particularly, he~atopoietic stem cells, with a gene transfer vector, partic~larly, a retrovirus vector; to resultant mammalian cells produced thereby; method of activatin~ stem cells; method of adoptive transfer of said cells; medicament compositions comprising said ells, methods of ~edical tre~tment u~ing said medicament~, and method~ of gen~ transfer in living organisms for non-medical purposes.
B~GRO~D TO ~Xg I~Vg~IO~
Gene tran~fer technology provide~ a powerful approach to the modification and study of cellular function. Efficient gen.e transfer into the genetic material of living organis~ i5 beao~ing increasingly desira*le and gaining importance. Transfer into plant c~lls, either for dissase control or to add or i~prove a desirable characteristic, is well establi~hed. In the medical field specific applications include the treatment ~ :
of genetic disease,~ cancer therapy, 1~5~6 and characterization of the developmental program of stem 2 ~
cells.7 A list of conditions that ara candidates for gene therapy comprises thalassemias, ADA de~iciency, PNP
deficiency, CGD, lysosomal storage! diseases, leukemias and other cancers~ myelodysplastic syndromes, viral infections, vitamin deficiencies, inborn errors of metabolism, infections and suppression or enhancement of normal functions post therapies for other illne~.
The target in most o these applications is the pluripotent hematopoietic stem cell, but the low frequency and quie~cent nature o~ stem cell~ neces~itates a highly ef~icient gene transfer syst2m. To date, retroviruses have been the most useful vector~ for gene transfer.l~4~8 Foreign genes hav~ been introduced in YitrO
into hematopoietic progenitors of many specie~ including the mouse, ~15 dQg,1~21 nonhuman primate~ ~ and hu~ans.~S~
28 Gene transfer and expression into pluripotent stem cells capable of long-ter~ reconstitution has only been shown ~or the mouse, using a variety of vectors.~ S
Despite these sucsesses, major issues remain to be resolved before therapeutic measures using gene transfer into human hematopoietic cell~ can be considered feasible.
One of these issues is the need to develop methods to transfer genes of interest at high ef~iciency into stem cells capable o~ reconstituting the hematopoietic system o~ large animals. In general, marrow cells infected in vitro have noS been maintained at high levels in vivo after autologous transplantation. Thi~ result has been attributed to the inability to infect a sufficiently large n~mber o~ primitive stem cells with reconstituting capacity.t~26 Mo~t infection protocols involve short-t~erm (1 to 3 day~) cocultivation of BM
cells with virus-containing media, or cocultivation over irradiated monolayers of virus-producing fibroblasts (reviewe~ by Kohn et al4~. Evidenca indicates that cell .. . . . .
- 2~8~4~
- 3 ~ SL276 cycling may be necessary for retroviral reverse transcription and integration, 1~27~28 and this ~uggests that a possible rea~on for the failure o~ retroviral vectors to transduce foreign DNA inko mammalian hematopoietic stem cells may be that they are noncycling in unmanipulated marrowO Further, there is a requirement that genetically altered ste~ cells undergo substantial proliferation in vivo for their presence to be detected.
Such activation o~ genetically alt~red stem cells has not been achieved thus far, except for the present work.
One prereguisite for clinically relevant retrovirally mediated gene transfer into hematopoietic cells is the need to achieve highly efficient infection of reconsti~uting cells.~467 LTMCs can provide acce.~s to pluripotent ctem cell~ over an extended period of time in a containsd environment, and therefore provide theoretical advantage3 in retroviral infection protocols.
In LTMCs it may be pos~ible to create lnLyi~Q conditions that result in the activation of normally quiescent he~atopoietic stem cells to cell cycling statu~, thereby making them amenable to retrovirally-mediated genz transfer while simultaneou~ly enhancing th~ chances that genetically ~anipulated stem cells would remain actively cycling in vivo.
Previous attempts to achieve gene transfer into ~TMC
cells have involved exposures to re~rovirus before long-ter~ ~ulture, 25~ or in~ection of LT~C maintained for only 6 days.20 Hematopoietic growth factor~ have been used to augment in vitro gene transfer into hematopoietic cells,~ and preexisting adherent layer~ have been used to enhance culture viability~20 A ~urther prerequisite ~or clinical implementation of retroviral ~ediated genQ transfer into hematopoietic cells in LT~Cs :is acceptable toxicity. 1'4 Cocultivation and selection have been used to enhance gene trans~er 2~8~
Such activation o~ genetically alt~red stem cells has not been achieved thus far, except for the present work.
One prereguisite for clinically relevant retrovirally mediated gene transfer into hematopoietic cells is the need to achieve highly efficient infection of reconsti~uting cells.~467 LTMCs can provide acce.~s to pluripotent ctem cell~ over an extended period of time in a containsd environment, and therefore provide theoretical advantage3 in retroviral infection protocols.
In LTMCs it may be pos~ible to create lnLyi~Q conditions that result in the activation of normally quiescent he~atopoietic stem cells to cell cycling statu~, thereby making them amenable to retrovirally-mediated genz transfer while simultaneou~ly enhancing th~ chances that genetically ~anipulated stem cells would remain actively cycling in vivo.
Previous attempts to achieve gene transfer into ~TMC
cells have involved exposures to re~rovirus before long-ter~ ~ulture, 25~ or in~ection of LT~C maintained for only 6 days.20 Hematopoietic growth factor~ have been used to augment in vitro gene transfer into hematopoietic cells,~ and preexisting adherent layer~ have been used to enhance culture viability~20 A ~urther prerequisite ~or clinical implementation of retroviral ~ediated genQ transfer into hematopoietic cells in LT~Cs :is acceptable toxicity. 1'4 Cocultivation and selection have been used to enhance gene trans~er 2~8~
eficiency,4~8~ but result in significant in vitro toxicity that compromises the growth of LT~Cs and negates som~ of the advantages of using progenitors in LT~Cs as targets for gene transfer.
Experiments in the mouse~ 3~have clearly shown that incubation of BM with hematopoietic growth factors be~ore infection increases gene trans~er efficiency, pre~mably by stimulating the stem cells into cycle. S~milar approaches using human progenitor!~ have re~ulted in a fivefold to 10 fold increase in gene transfer efficiency.~32 One of the potential problemQ with these short-term in vitro in~ection schema i~ the los3 of stem cells. This is especially true for procedures that involve cocultivation over irradiated viru~ producing fibroblasts in which stem cells,~which have adherent properties, 33 may be lost on th~ f ibroblast~.
It is known that retroviral infection co~bined with Dexter type long-term marrow culture (LTMC) offers opportunities for improved gene transfer through 20^ manipulation of marrow proqenitor c~lls.~~
R~ B~C~ ~18T
The present specification refsrs to the following publications, each of which is expressly incorporated by reference hsrein.
P~ICA~I0~8 :~
1. ~eatherall DJ: Gene Therapy in perspective.
Nature 349:275, 1991.
2. Feigner PL, Rhodes G: Gene therapeutics.
Nature 349:351, 1991.
3. Beutler E, Sorge J: Gene transfer in the treatment of hematologic disease. Exp Hematol 18:857, 1990.
4. Kohn DB, Anderson WF, Blaese RM: Gene therapy for ,, .
:
2~ 3~
_ 5 _ SL276 genetic diseases. Cancer Invest 7:179, 19$9.
Experiments in the mouse~ 3~have clearly shown that incubation of BM with hematopoietic growth factors be~ore infection increases gene trans~er efficiency, pre~mably by stimulating the stem cells into cycle. S~milar approaches using human progenitor!~ have re~ulted in a fivefold to 10 fold increase in gene transfer efficiency.~32 One of the potential problemQ with these short-term in vitro in~ection schema i~ the los3 of stem cells. This is especially true for procedures that involve cocultivation over irradiated viru~ producing fibroblasts in which stem cells,~which have adherent properties, 33 may be lost on th~ f ibroblast~.
It is known that retroviral infection co~bined with Dexter type long-term marrow culture (LTMC) offers opportunities for improved gene transfer through 20^ manipulation of marrow proqenitor c~lls.~~
R~ B~C~ ~18T
The present specification refsrs to the following publications, each of which is expressly incorporated by reference hsrein.
P~ICA~I0~8 :~
1. ~eatherall DJ: Gene Therapy in perspective.
Nature 349:275, 1991.
2. Feigner PL, Rhodes G: Gene therapeutics.
Nature 349:351, 1991.
3. Beutler E, Sorge J: Gene transfer in the treatment of hematologic disease. Exp Hematol 18:857, 1990.
4. Kohn DB, Anderson WF, Blaese RM: Gene therapy for ,, .
:
2~ 3~
_ 5 _ SL276 genetic diseases. Cancer Invest 7:179, 19$9.
5. Rosenberg SA, Aebersold P. Cornetta K. Rasid A, Morgan RA, Moen R, Karson EM, Lotze MT, Yang JC, Topalian SL, Merino MJ, Culver K, Miller AD, Blaese 5RM, Anderson WF~ Gene transfer into hu~ans-Immunotherapy of patient~ with advanced m~lanoma, using tumor-infiltrating lymphocytes modi~ied by retroviral gene tran~duction. N Engl J M~d 323:601, 1900 .
106. Friedmann T: Progress toward human gene therapy.
Science 244:1275, 1989.
7. Bogg~ SS: Targeted gene modification for gen~
therapy of stem cells. Int J Cell Cloning 8:80, 1990 .
158. Dick JE: Retrovirus-mediated gene tran~er into hematopoietic stem cells. Ann NY Acad Sci 507:242, 1987.
9. Dzierzak EA, Papayannopoulou T, Mulligan RC:
Lineage-speci~ic expres~ion of a human B~globin gene 20in murine bone marrow transplant recipients reconstitutedwith retrovirus-tran~duced ~tem cells.
Nature 331:35, 1988.
10. Bender MA, G~linas RE, Miller AD: A majority of mice show long-term expres~ion o~ a hu~an B-globin 25gene after retrov$rus tran~f~r into hematopoietic stem cells. Mol Cell Biol 9:1426, 19890 11. Li~ B, Apperley JF, Orkin SH, Williams DA: Long-term expression of hu~an adeno~ine deaminase in ~ice tran~planted with retrovirus~in~cted hematopoietic 30s~em cellx. Proc Natl Acad Sci USA 86:~892, 1989.
12~ Szilvassy SJ, Fraser CC, Eaves CJ, Lansdorp PM, Eave~ ACo ~Iu~phrie~ RK: Retrovirus mediated gene transfer to puriied hematopoietic stem c~ with long-term lympho-myelopoietic repopulating ability.
35Proc Natl Acad Sci USA 86:8798, 1989.
:
- . ~ :.
- `
106. Friedmann T: Progress toward human gene therapy.
Science 244:1275, 1989.
7. Bogg~ SS: Targeted gene modification for gen~
therapy of stem cells. Int J Cell Cloning 8:80, 1990 .
158. Dick JE: Retrovirus-mediated gene tran~er into hematopoietic stem cells. Ann NY Acad Sci 507:242, 1987.
9. Dzierzak EA, Papayannopoulou T, Mulligan RC:
Lineage-speci~ic expres~ion of a human B~globin gene 20in murine bone marrow transplant recipients reconstitutedwith retrovirus-tran~duced ~tem cells.
Nature 331:35, 1988.
10. Bender MA, G~linas RE, Miller AD: A majority of mice show long-term expres~ion o~ a hu~an B-globin 25gene after retrov$rus tran~f~r into hematopoietic stem cells. Mol Cell Biol 9:1426, 19890 11. Li~ B, Apperley JF, Orkin SH, Williams DA: Long-term expression of hu~an adeno~ine deaminase in ~ice tran~planted with retrovirus~in~cted hematopoietic 30s~em cellx. Proc Natl Acad Sci USA 86:~892, 1989.
12~ Szilvassy SJ, Fraser CC, Eaves CJ, Lansdorp PM, Eave~ ACo ~Iu~phrie~ RK: Retrovirus mediated gene transfer to puriied hematopoietic stem c~ with long-term lympho-myelopoietic repopulating ability.
35Proc Natl Acad Sci USA 86:8798, 1989.
:
- . ~ :.
- `
13. Eraser CC:, Eaves C~, Szilvassy SJ, Humphries ~
Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells . Blood 76 :1071, 1990 .
14. Capel B, Hawley RG, Mintz B: Long-and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers.
Blood 75:2267, 1990.
15. Van Zant G, Chen JJ, Scott-Micus K: Developmental potential of hematopoietic stem cells determined using retrovirally marked allophenic marrow. Blood 77:756, 1991.
16. Kwok WW, Schuening F, Stead RB, Miller AD:
Retroviral transfer of genes into canine hematopoietic progenitor cells~in culture: A model for human gene therapy. Proc Natl Acad Sci USA
83:1, 1986.
17. Eglitis MA, Kantoff PW, Jolly JD, Jones JB, ~nderson WF, Lothrop Cd: Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using ratroviral vec~ors. ~lood 71:717, 1988.
18. Stead R3, Xwok WW, Storb R, ~iller AD: Canine model ~or gene therapy: Inefficient gene axpres~ion in dogs reconstituted with autologous marrow infected with retroviral vector~. Blood 71:742, 1988.
19. Schuening FG, Storb R, ~ash R, Stead RB, Kwok WW, Miller AD: Retroviral transfer of gene3 into canine hematopoietic progenitor cellsO Adv Exp Med Biol 241:9, 198~
io. Schuening ~G, Storb R, Stead RB, Goehle S, Nash R, Miller AD: Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term l~arrow culture. Blood 74:1S2, 1989.
21. Al-Lebban ZS, Henry JM, Hones JB, Eglitis MA, Anderson W:F, Lothrop CD: Increased efficiency of gene tr~nsfer with retroviral vectors in neonatal hematopoietic prsgenitor cells. Exp. Hematol 18:180, l99o.
22. Kantoff PW, Gillio AP, McLachlin JR, Bordignon C, Eglitis MA, Kernan NA, Moen RC, Kohn DB, Yu S~, Karson E, Karlsson S, Zwiebel JA, Gilboa E, Blaese RM, Nienhuis A, O'Reilly RJ, Anderson WF:
Expression of human adenosine deaminase in nonhuman primates after retroviru~-mediated gene transfer.
J. ~xp ~ed 166:219, 1987.
23. Bodine DM, McDonagh KT, ~randt SJ, Ney PA, Agricola B, ~yrne E, Nienhuis ~W: Development of a high-- titer retrovirus producer cell line capable of gene tra~sfer into rhesu~ monkey hematopoietic stem cells. Proc Natl Ac~d Sci USA 87:3738, 1990O .
24. Laneuville P, Chang W, Xamel-Reid S, Fauser ~A, Dick JE: High-efficiency gene transfer and e~pression in normal human hematopoietic cells with retrovirus vectors. Blood 71:811, 1988.
25. Hughes PFD, Eaves CJ, Hogge DE, Humphrie~ RK: High-efficiency gene trans~er to hu~an hematopoietic cells maintain~d in long-term marrow culture. Blood 74:1915, 1989.
2~. Bordigno~ C, Yu SF, Smith CA, Hantzopoulo~ P, Ungers GE, Keever CA, O'Reilly RJ, Gilboa E. Retroviral vector-mediated high-e~ficiency e~pression of adenosine deaminase (ADA3 in hematopoietic long-term cultures of ADA-de~icient marrow cslls. Proc Natl A~ad Sci USA 86:6748, 1989.
27. S~oeckert C~, Nicolaides NC, Haines RM, Surrey S, Bayever E- Retroviral transfer of gene3 into erythroid progenitors derived from human peripheral blood. E~p. Hematol 18:1164, 1990.
28. Wieder R, Cornetta K, Kessler SW, Anderson FW:
Increased efficiency of retroviral-mediated gene 2~84~
Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells . Blood 76 :1071, 1990 .
14. Capel B, Hawley RG, Mintz B: Long-and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers.
Blood 75:2267, 1990.
15. Van Zant G, Chen JJ, Scott-Micus K: Developmental potential of hematopoietic stem cells determined using retrovirally marked allophenic marrow. Blood 77:756, 1991.
16. Kwok WW, Schuening F, Stead RB, Miller AD:
Retroviral transfer of genes into canine hematopoietic progenitor cells~in culture: A model for human gene therapy. Proc Natl Acad Sci USA
83:1, 1986.
17. Eglitis MA, Kantoff PW, Jolly JD, Jones JB, ~nderson WF, Lothrop Cd: Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using ratroviral vec~ors. ~lood 71:717, 1988.
18. Stead R3, Xwok WW, Storb R, ~iller AD: Canine model ~or gene therapy: Inefficient gene axpres~ion in dogs reconstituted with autologous marrow infected with retroviral vector~. Blood 71:742, 1988.
19. Schuening FG, Storb R, ~ash R, Stead RB, Kwok WW, Miller AD: Retroviral transfer of gene3 into canine hematopoietic progenitor cellsO Adv Exp Med Biol 241:9, 198~
io. Schuening ~G, Storb R, Stead RB, Goehle S, Nash R, Miller AD: Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term l~arrow culture. Blood 74:1S2, 1989.
21. Al-Lebban ZS, Henry JM, Hones JB, Eglitis MA, Anderson W:F, Lothrop CD: Increased efficiency of gene tr~nsfer with retroviral vectors in neonatal hematopoietic prsgenitor cells. Exp. Hematol 18:180, l99o.
22. Kantoff PW, Gillio AP, McLachlin JR, Bordignon C, Eglitis MA, Kernan NA, Moen RC, Kohn DB, Yu S~, Karson E, Karlsson S, Zwiebel JA, Gilboa E, Blaese RM, Nienhuis A, O'Reilly RJ, Anderson WF:
Expression of human adenosine deaminase in nonhuman primates after retroviru~-mediated gene transfer.
J. ~xp ~ed 166:219, 1987.
23. Bodine DM, McDonagh KT, ~randt SJ, Ney PA, Agricola B, ~yrne E, Nienhuis ~W: Development of a high-- titer retrovirus producer cell line capable of gene tra~sfer into rhesu~ monkey hematopoietic stem cells. Proc Natl Ac~d Sci USA 87:3738, 1990O .
24. Laneuville P, Chang W, Xamel-Reid S, Fauser ~A, Dick JE: High-efficiency gene transfer and e~pression in normal human hematopoietic cells with retrovirus vectors. Blood 71:811, 1988.
25. Hughes PFD, Eaves CJ, Hogge DE, Humphrie~ RK: High-efficiency gene trans~er to hu~an hematopoietic cells maintain~d in long-term marrow culture. Blood 74:1915, 1989.
2~. Bordigno~ C, Yu SF, Smith CA, Hantzopoulo~ P, Ungers GE, Keever CA, O'Reilly RJ, Gilboa E. Retroviral vector-mediated high-e~ficiency e~pression of adenosine deaminase (ADA3 in hematopoietic long-term cultures of ADA-de~icient marrow cslls. Proc Natl A~ad Sci USA 86:6748, 1989.
27. S~oeckert C~, Nicolaides NC, Haines RM, Surrey S, Bayever E- Retroviral transfer of gene3 into erythroid progenitors derived from human peripheral blood. E~p. Hematol 18:1164, 1990.
28. Wieder R, Cornetta K, Kessler SW, Anderson FW:
Increased efficiency of retroviral-mediated gene 2~84~
transfer and expression in primate bone marrow progenitors after 5-f luorouracil-induced hematopoietic suppre~sion and recovery. Blood 77: 448, 1991 .
29. Ekhterae D, Crumbleholme T, Karson E, Harrison MR, Anderson WF, Zanjani ED Retroviral vector-mediated transfer o~ the bacterial neo~ycin resistance gene into fetal and adult sheep and human hematopoietic progenitors in vitro. Blood 75:365, 1990.
30. Bodine DM, Karls~on S, Nienhui~ AW Co~bination of interleukins 3 and 5 preserv2s st m cell function in culture and ~nhances retrovirus-medi~ted gene transfer into he~atopoietic ste~ cells. Proc Natl Acad Sci USA 86:8897~ 19~9.
lS 31. Fletcher F, Williams D, Mali~w~ki C, Anderson D, Rives M, Belmont J: ~urine leuX mia inhibitory factor enhances r~troviral-vector infection efficiency of hematopoietic progenitors. Blood 7Ç 11~98, l990o 32. Dick JE, Kamel Reid S, Murdoch B, Doeden~ M: Gene transfer into normal human hematopoietic cells u ing in vitro and in vivo as~ays. Blood 78:624, 1991.
33. Gordon ~Y Adhe~ive properties o~ haemopoietic stem cells. Br J Haematol 68:149, 1988.
34. Toksoez D, Dexter TM, Lord BI, Wright EG, Lajtha LG:
The regulation of hemopoe~is in long-term bon~
marrow culture II. Stimulation and inhibition of stem cell proliferat~on. Blood 44:931, 1980.
35. Cashman J, Eaves AC, Eaves CJ: Regulated proliferation of primitive hematopoietic progenitor cell~ in long-term human marrow cultures. Blood 66:1002, 1985.
36. Carter RF, Kruth SA, Valli VE0, Dub~ ID: Long-term culture of canine marrow: Cytogenetic evaluation of purging of lymphoma and leukemia. Exp. Hematol -'` 2~8~
- g SL276 18:995, 1990.
37. Armentano ~, Yu 5, Kantoff PW, von Ruden T, Anderson WF, Gilboa E: Effect of internal virus sequences on the utility o~ retroviral vectors. J Virol 61:1647, 1987.
38. Saiki RK, Scharf St Faloona F, Mulli~ KB, Horn GT, Erlich H~, Arnheim N: Enzy~atic amplifi~ation of B~
globin genomic sequences and restriction ~ite analysis for diagnosis of sickle cell anemia.
Science 230:1350t 1985.
39. Abrams-Ogg ACG, Kruth SA, Carter RF, Valli VEO, DUba ID, Dick JE: Autologou~ bone marrow transplantation u~ing long term bone marrow culture in dog~. J Vet Intern Med 4~130, 1990 (abstr).
40. Storb R, Raf~ RF, Appelbaum FR, Graham TC, Schuening FG, Sale G, Pepe M- Compari~on of ~ractionated to single-dose total body irradiation in conditioning canine littermates for DL~identifical marrow grafts. Blood 74:1139, 1989.
41. Kalebo M. Garcia JV, Osborne WA, ~iller AD:
Expression of hu~an adeno~ine dea~inase in mice after tran~plantation of genetically modified bone marrow. ~lood 75:1733, 1990.
42. Joyner A, Keller G, Phillip~ R~, Bern3tein A:
Retroviru~ transfer o~ a bacterial gene into mouse hematopoietic progenitor cells. Natur~ 305:556, 19~3.
43. Storb R, Raff R~, .Appelbau~ FR, Schuening ~G, San~maier B~, Graham TC, Thomas ED What radiation c~o~e ~or DL~-identifical canine marrow grafts?
Blood 72:1300, 1988; erratum Blood 73:624, 1989.
44. Down JD., Tarbell NJ, Thames HD, Mau~h P~: Syngeneic an~ allogeneic bone marrow engraftment a~ter total body irradiatio~: Dependencs on dose, does rate, and fractionation. Blood 77:661, 1991.
2 ~
.
45. Mic~lem HS, Ford CE, Evan~ EP, Ogden DA: :
Compartmen~s and cell ~low~ within the mouse haemopoietic system. I. Re.stricted interchange between haemopoietic site~. Cell Tissue Kinet 8:219, 1~75.
46. Micklem HS, Ogden DA, Evans EP, Ford CE, ~ray JG:
Compartments and cell flow~s within the mouse haemopoietic system. II. Estimated rates of interchange. C~ll Tissue Kinlet 8:233, 1975.
~, ~U~ Y 0~ T~ INV~N~IO~
It is a~ object of the present invention to provide an improved protocol for gen transfer into receptive targPt cells, target cells for use in enhancement of the genetic material of living organi~ms, such a~
microoryanisms, ~ungi, pl2nts and animals, including stem cells and target c~118 for transient expression, It is a further object of the present invention to provide desired gene-containing target cells and compo~ition~ of u5e in medica~ent~ ~or ma~al~ and methods o~ treatmen~ o~ the ~mmal3 using ~aid cells.
It is a yet further object of the invention to provide a method ~or activating stem cells and a method for the adoptiv~ tran~fer o~ ~aid cells.
Thu~; the method according to the invention compri6es subjecting cells in long term culture to repeated exposure to ~ene transfer vahicle~, particularly retroviru~ vector, preferably ti~ed to coincide with the wave o~ cellular proliferation thought to occur a~ter media replanishment. While not being bound by theory, it is bslieved that the method accordlng to the invention allows stem cel:Ls maintained in culture to be triggered -' 2~8~
into activated cell-cyle status thereby providing the opportunity for optimizing retroviral infection over a relatively long period of time while simultaneously creating a population of activated stem cells with the potential ~or extensive proliferat.ion n vivo.
Accordingly, in its broadest a~pect the invention provides a method o~ effecting trans~er of a gene into a gene tran~fer target cell comprising ~tabli~hing a long ter~ culture containing said cell~ in a uitable medium and exposing said culture to multiple, periodic infection~ of a gene transfer vehicle containing said gene to provide said gene - containing target cell.
Preferably, the media is periodically, partially substituted with fresh media.
15 The vector is provided in an amount effective to transfer the gene into the cells, and provided at the steps o~ development o~ the cells such that the cell~ are capable of being in~ected by the vector and infusion of marked ~te~ cells without conditioning host.
While of general applicability for ef~icient gene trans~er in receptive target cell sys~em~, the met~od of the in~ention is of particular value in the production of long term marrow culture cells genetically marked by retroviral vectors and infusion of marked ~tem cells without conditioning of host.
The methods according to the invention are applicable to all receptive target cell~, particularly stem cells such as epithelial stem cells, exocrine gland stem cells, endocrine gland stem cells, stromal stem cells,- long term culture initiating cells,-hepatocytQ
stem cells, pancreatic stem cells, neuroendocrine stem cells, connective tissue ~tem cells, ~ibroblast stem cells, mesench~nal stem cells, adipose cell tem cells, mammary gland stem cells, reticuloendothelial stem cells, lipid ste~ cells, chondrocyte stem cells, oste4progeni~0r 2 0 ~
- 12 ~ SL276 stem cells, osteocyte stem cells, muscle fiber stem cells, neuronal stem cells, epidermal stem cells, keratinocyte stem cells, Langerhans stem cells, m~lanocyte stem cells, sebaceous gland ste~ cells, sweat gland stem cells, mucous stem cells, serous cell stem :
cells, odontoblast stem cells, islet of Langerhans stem cells, alveolar stem cells and ret:inal stem cells.
In addition, the invention i~ of value in providing modifications to target cells with less cap~city ~o~ cell renewal by modifying the duration of culture, the retroviral titer, the retrovirus, timing and durakion of exposure to gene transfer vehicle, and other parameter3, it is possible to specifically target cell~ that have specific capacities for proliferation. Thi~ approach will have applications in gene ~therapy for somatic disease where transient expres~ion of the gene is a desirable outcome. Target cell~ for trancient expression include mast cell~, eosinophils, plasma cells, lymphocyte~, monoblast~, promonocyte~, monocytes, macrophages, basophils neutrophil~, erythrocytes, granulocytes, megakaryocytes, platelets, osteoblasts, osteocyte~, osteocla~ts, ~ibroblast~, epithelial cells, stromal cells, hepatocytes, pancreatic cells, neuroendocrine cell~, connective ti~sue c 11R, mesenchy~al cell~, adipocytes, mammary gland cells, reticuloendothelial cells, lipid cells, chondrocytes, muscle cells, neuron~, epidermal cells, ~angerhans cells~
melanocytes, gland cells,. mucous cells, serous cells, odontobla~ts, i~let of Langerhans cells, alveolar cells and re~inal cells.
Pref erably, the method as hereinabove de~ined comprises establishing said culture in a first suitable medium; exposinl~ said culture to a first infection of said gene transfer vehicle; including said infected cultùre for a sufficient period o~ time to ef~ect cell 2~8~44 proliferation; substituting a portion of said firs~
suitable medium with a second ~uitable medium to provid2 a first resultant medium; exposing said culture to a second infection of said gene transfer vehicle; further incubating said inf~cted culture for a sufficient period of time to e~fect ~urthe~ cell proliferation and in vitro cell renewal; and recovering said gene-containing target cell as w~ll.
More preferably, the method f~lrther co~pri es prior to recovering said gene-containing target cell, substituting a portion of said ~irst resultant mediu~
with a third suitable medium to provide a ~econd resultant medium; exposing said culture to a third infection of said gene transfer vehicle; and further incubating said culture for a sufficient period of time to effect yet further cell prolif~ration and in vitro cell renewal.
The gene-trans~er vehicle may be added simultaneously to the cultures with the exchanging ~edia, but, preferably, each mediu~ i~ added a few hour~ be~ore, pre~erably, say, 10 hours bs~ore or more preferably the day before the gene-transfer vehicle is added to the resultant culture.
The cultures are ~llowed to incubate ~or sufficient periods of time to effect cell pxoliferation, typically at least ~or 3 days but preferably at least 7 days.
These significant period~ of time for incubation in an environment that ~imulates in vivo microenvironment allow~the stem cells to proliferate and di~ferentiate by activa~ion and lenhancement o~ the ~elf-renewal ~yste~.
Multiple, partial renewal of the media, periodically, encourages the progenitor cells to address ~he shock to the cell system by 3timulating activation o~ the entire progenitor cell hierarchy available for qene-transfer.
~S8~
It is widely beli~ved that in vivo, microenvironment~ that normally support the proliferation and differentiation of stem cells must be appropriat~ly conditioned if they are to receive and support 'the proliferation of exogenously administered stem cells.
Thus, many investigators elect to subject the recipients o~ genetically manipulated hematopo:ietic cells to marrow ablative conditioning prior to adoptiv~ gene tran~ferO
The assumption is that the he~atopoietic cells provided post-marrow ablation will home to the "empty'i marrow microenvironment, theoretically ideally suited for their maintenanc~.
Thexe is evidenc~, however, that in large mammals the maximum death-sparing level of irradiation ic not totally marro~ablative. If animal~ subjected to such irradiation are vigorou~ly supported during the pancytopenic post irradiation phase, their hematopoietic system~ eventually recover. During this recovery phase, the animals' hematopoietic systems are most likely subjected to self-regulatory feedbacX control mechanisms that strongly support the proliferatio~ of thos~ stem cells would presumably face strong co~petition from their endogenou~ counterparts.
We hav~ di~coversd that in vivo m~intenance of ~B
yitro activated hematopoietic ~tem c~113 have been signi~icantly enhanced in recipient3 that wera not subjected to a~y type o~ marrow conditioning. LTMCs fro~
15 dog3 were established and subjected to gene transfer using 21-day, 3 cycle transduction protocol with th~ N2 -30 ratroviral vector. The dogs were divided in~o 3 groups of 5 each. ~ach group received a different number of infu~ed LTMC cells. No dogs underwent marrow conditioning. Dog~ were followed a~ before for evaluation of the contribution mad~ to hematopoie~i~ by the LTMC cQll~.
8 ~ ~
- l5 - SL276 Accordingly, in a further aspect the inven ion provides a method of enhancing 1:he proliferation o~
pluripotent stem cells in vivo in a mammal comprising administering activated stem cells a~ hereinabove defined to said mammal and allowing proli~eration of said c~
in said mammal in the absence of ablative marrow conditioning.
By the term "ablative marrow conditioning" is meant the removal of all host cells from a particular site in thq recipient, which c~lls include abnormal and nor~al tissue. Such abnormal cells ganerally comprise gen~tically defectiv~, genetically deficient, mutated, cancerous and virally in~ected cells. Ablative marrow conditioning include~ chemotherapy and irradiation treatments.
In yet a further a~pectV the invention provides a method ~or activating quiescent repopulating stem cells in vitro to provide activated stem cells which maintain their activity in vivo comprising establishing a long term culture containing said quiescent ~tem cell~ in a suitable m~dium and m~ltiple, periodic partial substitution o~ s~id medium to provide said activated stem cell and recovering said activated ste~ cell.
PreferablyO the method comprise~ establishing a method as h~reinabove defined compri ing es~ablishing said culture in a ~irst suitable medium; incubating said culture for a first su~ficient period of time to ef~ect cell proliferation and. ~ vitro cell renewal;
substituting a portion of said fir~t suitable medium with a second suitable medium to provide a ~irs~ resultant medium; further incubating ~aid culture for a second sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said actlvated stem Cell7 ':
: ' ~ ' . ' ' .
'' ''" ' ~
2~8~84~
The pre~erred periods of time to e~fect cell proliferation is at least 3 days.
D I~D ~8CRIP~I0~ OP ~ IN~I0 In order that the invention may be better under~tood a preferred embodiment will now be described, by way of example only, with reference to the accompanying drawings, wherein:
Figure 1 represents a bar chart summarizi~g the numbers of cells and CFU-GM harvested ~rom nine dog~;
Figure 2 represents ~A) a titration curve ~or G418 resistance; and (B) agarose gel electrophores~3 of neo~
a~plified sequence~ from dog 1;
Figure 3 represents southern analysis o~ neo-amplified sequences in LT~C adherent layer cells from dog 3;
Figure 4 repre~ent~ agarose gel electrophoresiR o~ ~eo-ampli~ied sequences in peripheral blood cells obtained from dogs 2, 3 and 5 a~ter transplantation;
Figura 5 represents southern analysis neo-amplified seguence~ in marrow aspirated from dog 2 at 13 months posttransplantation; and Figure 6 repre~ents agarose gel electrophone~i~ of neo-amplified sequenceso individually plucked colonies obtained fro~ dog 3 at 8 month~ posttransplant.
Figure 7 represent~, as Figure 7a, Figure 7b and Figure 7c percentages of .G418 resistant marrow-derived hematQpoietic progenitors for several group~ of dogs.
~ong term marrow culture from normal dogs w~s exposed to ~hree round~ o~ infection with neo~containing retrovirus produced by the packaging cell line PA317-N2 during twenty-ona day~ of culture. The results showed that the nQomycin phosphotransferase gene (neo) wa~
transferred with high efficiency into hema~opoietic ~ o ~
progenitors in LTMCs. After infusion of infect~d LTMC
cells into autologous recipients, were detected progency cells of genetically marked marrow progenitors, with and without the use of marrow condit-ioning by total body S irradiation ~TBI~.
~A~R~A~ A~D ~T~OD8 Normal dogs were obtained ~rom the Central An~mal Facility of the University of Guelph and maintained in the Veterinary Teaching Hospital until they recovered from all procedures. All protocols were approved by committee~ of the University of Toronto. The Toronto Hospital f and the University of Guelph concerned with animal care, biohazards, and ethical review.
p~pARa~rIo~ o~ VI~ $QP13R~ 8 The necl retroviral vector used wa. the Moloney murlne leukemia-derived vector, N2,~ 8e~ore ~arrow harvest~ the help~r cell line PA317.N2 was grown in a minimal essential medium (alpha-Me~) media containing 4.5 g/L l-glutamine, 10% fetal bovine serum, and 0.2g/L G418.
The day before marrow harvest, the alpha-~EM media was replaced by media used ~or LTMCs . 36 On the day of the marrow harvest, this media was collected and a cell free supèrnatant co~taining -1 x 106 viruslmL ln long-term culture media was prepared by centrifugation at 400g for 15 minutes. Ali~uot~ o~ viru~-containing media were similarly prepared for second and third infection~ of each culture. No helper viru~ was detected by a sensitive viral rescue assay in the~e viral ~upernatants or from th~ ~T~C~ after 21 day . .
2Q8~
GEN~ TRaN~FBR INTO C~NINB ~C
After induction o~ general anesthesia, marrow and blood cells were aspirated from the iliac crests, ischiatic crests, and proximal femori and humeri o~ each dog, and placed directly into preservative ~ree heparin (2,000 U/lOOmL of aspirated marrow). After determination o~ the total viable nucleated cell count ~y trypan blue exclusion, the mononuclear cell fras:tion was prepared by density centri~ugation and LT~Cs were initiated as prepared by density centrifugation and LTMCs were initiated as previously described 36 along with the following modifications: each marrow culture was set up in culture m~dium containing retrovirus and exposed to virus on two more occasion~ during the culture period, with the last exposure to virus occurriny 6 day~ be~ore culture harvest and in~usion at 21 days.
Specifically, cultures were initiated by inoculatillg Corning T-150 ~lasks (Corning, Corning, NY) with 1~2 x 1o8 mono~uclear cells in 60 mL of media containing virus.
2 0 On days 7 and 14 of incubation, thQ f lask wQre demi-depopulated and fed with non-virus containing media. On days 8 and 15, on half of the supernatant layer of each flask was removed, the cells were pe.lleted and resuspended in fresh media containing ~1 x 106 virus/mL.
A~ter 21 day3 of culture, all adherent cells were recovered by trypsinization, washed three times in presexvative free, pH indicator free Hank's buffered saline solution (GIBCO, Grand I~land, NY), and kept at room temperatur~ for 1 to 3 hours before bolu -injection b~ syringe and butterfly needle infusion set into the cephalic vein of the autologous recipient.
. , . . . ~ .~ . ,;
2~84~
PRo~ITO~ A88~Y~
Progenitor cells (colony ~o~ming unit granulocyte macrophage [CFU-GM], burst fvrming unit erythroid [BFU-E], and CFU-~ix) were a~sayed, at least in duplicate, in S 1 mh of methylcellulose media plated in 35 mm petri dishes as previously described.36 Resistance to the ~eomycin analog, G418, was assess;ed by counting the numbers of colonies ari~ing by day 10 in plates containing 1 mg/m$ G418 comp~red with plates containing no added G418. For evaluation o~ T cells, nucleated peripheral blood cells were maintained in liguid culture consisting RPMI 1640 IGIBC0), 10% fetal calf serum (FCS), 1% L glutamine, and 1~ pen/strep, at 106 cells /mL.
Phytohemagglutinin (PMA; Wellcome, Dartford, U.K.) was added for 72 hours at culture initiation after which time cells were pelleted by centri~ugation and resuspended in fresh media supplemented with interleukin 2 (Cetus, Norwalk, CT) at 20 U/m~ twice weekly ~or 2 to 3 w~eks.
DBT~C~Io~ o~ o ~Y PO~ 8~ c~aI~ REac~ C~) :
Approxi~ately 106 cells fro~ ~resh blood, BM, cultured T c~ , or LTMCs were lysed and DNA was extracted ~or PCR ampliPication. DNA from day 10 CFU-GM, growing in the absence o~ ~418 and estimated to contain at least 20 cells, was obtained for PCR amplification by placing plucked colonie~ dir~ctly in 19 ~ of extraction buffer (50 m~ol/L XC1, 10 mmol/L Tri~ HCl pH 8.3, 2.5 mmol/L ~GC12, 0.1 mg/mL 2~ gelatin, 0.45% nonidet P40, O.45% ~ween 20~ and incubating with 10 proteinase K for 2 hours at 55C. The neo-specifi¢ oligonucleotide primeers were a 21 mer to the sen~s strand (5' AAAG~ATCCATCATGG~CTGAT 3'), and a 20mer to the antisense strand (5' ATGt'TCTTCGTCCAGATCAT 3'). Neo specific sequence~ were amplified according to standard CPtus protocol38 from 250 ng to ~g extracted DNA or DN~ obtained 2 ~ 4 ~
by direct lysi~ oP plucked colonies. The denaturatlon temperature wz3 94C, the annealiny teMp~rature was 68C, and the extension temperature waR 72Co ~he amplified DNA was identifi~d a~ter electrophoresi~ in 1% agarose and 2~ NuSi~ve G~ agarOsQ (FMC b.ioproducts, Rockland, ME) by staining with ethidium bro~ide. Southern blot~
were prepared by transferring the DNA fro~ the gel to a nylon membrane (Hybond; Amersham, Oakville, ontario, Canada). Blot3 were probed with a 32p labelled neo fragment. Specificity wa~ deter~irled by autoradiography with Kodak X-omat ~ilm (Rochester, NY~ Control specimens included serial dilutions of PA317.N2 and/or HL60N2 (an HL60 cell line containing a neo vector), reagents snly, BM cells from uninfected nor~al dogs, and CFU G~ derived ~ro~ as3ays o~ uni~fect~d normal canine marrow. Samples were con~idered positiv~ i~ a band identical to the positive control wa detectabl~ by ethidium bromide staining of the gel. Southern blots were used to confirm that suspected positives actually were neo specific se~uences.
MARRO~ ÇO~DI~IO~ A~D ~a~RO~ TRa~A~A~IO~
Dogs received identical preparative and isolation protocol ; details o~ the prQparativ~, irradiation, and support protocols have been described elsewhere.39 Marrow conditioning wa~ administered to transplant dogs and control dogs by bilateral ex~osure to a single ~Co source; the total midline dose was 6 Gy or S Gy in a single- ~raction at dose rates fro~ 10 cGy/~in to 75 CGY/~rn. DOgR recei~ing transplants without marrow conditioning wer~ not irradiatad and wer~ not administered antibiotics. Follow up speci~ens of periphèral blood and BM (obkained under general anesthesia) wer~ obtained at intervals and assayed as 2 ~
.
- 21 ~ SL276 described above by PCR amplification for the presence o~
neo.
D~Æ~IO~ O~ I~F~C~O~ O YI~ AND ~ CA~ION
CONP~ R~TR~VI~8 I~ PhA8~A OR R~CO~ BD DO~8 The PA317-N2 cell line was re~established from frozen stocks every 6 weeks to pre!vent the inadvertent production of replication competent virus. Plasma ~rom reconstituted dogs wa tested for thQ presence of infectious neo viru~ using Rat 2 cell~ a~ an in~icator line. Dog plasma was diluted to 10~ with a-MEM
containing 8 ug/mL polybrene and incubated ~or 24 hours with 2 x 105 Rat 2 cells on 60 mm tissue culture plates~
Higher plasma concentration~ cau~ed Rat 2 cells to grow poorly. After 24 hours, ~resh a ME~ wit~ 400 ug/mL G418 was added to the plates. Th~ plates were incubated ~or 7 days a~d scored for ~418R colonie~. The positive control was the PA317-Na ce~l line.
The dog plasma was also tested for the presence of replication competent helper virus using a marker resuce assay.4~ The diluted plasma, alpha-ME~, and polybrene were incubated with NeoNP-l cells ~or 24 hour~. N~oNP-l is a 3T3 cell linQ in~ectQd with ths MLVNEO.1 retrovirus vecto~2. Any helper viru~ in the test sampl~ will spread throughout the culture and resuce the ~LVN~O.1 vector that can then b~ assayed on Rat 2 cell~ or human HOC-7 cell~. The NeoNP-1 cells were passaged and grown for at least 1 week to permit viru~ spread. This supernatant wa~ diluted fro~ 10 to 10-3 and incubated with Rat 2 cells-to as~ay for the rescued MLVNEO.l vector. The positive control was supernatan~ from a packaging cell line known to ma:ke helper virus and the negative control was supernatant fro~ the NeoNP-1 cell line.
2 ~
RE8U~T8 - Tables 1 and 2 summarize the procedures and results for nine dogi~. Four dogs received the entir~ transplant protocol including marrow condi.tloning ("~C~", dogi3 1 through 4). The first dog di~d due to a failure of support (~ulminant Streptococcal septicemia) at 18 dayis postirradiation, but dog~ 2, 3 and 4 survi~ed and were . evaluated for evidence of retenti.on of genet~cally ~arked ;. hematopoietic celli3. Thre~ dogs rQceived the entire protocol except that marrow conditioning was completely omitted ("MC-", dogs 5, 6, and ?). Dog3 ~ and 9 were controls that received the entire protocol except that no cells of any kind were infused after marrow conditioning;
others recovered ~rom pancytopenia at approximately 35 days but dog 9 died after a sever~- acute rQaction to a platelet transfusion at 36 days.
:.,:, . . ~ . ... . , - . , r~ 2 ~ 8 ~ 4 J . ~
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i ~ ~x I ~ x ~ J l ~ i o ~ ~ ~ E
i~ 3J~ ~t ~ i~
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24 - SL~76 NARRO~ ~A~V~8 AND_C~ R~COV~Y FRO~ C~L~ 8 Figure 1 summarizes the numbers of cells harvested and then obtained after 21 days of LTMC. The mean number of mononuclear cells initially harvested was 1.6 x lo8 (range, 3.1 x 107 to 2.3 x 108) per kilogram of body weight (BW), which corresponded to a mean o~ 1.2 x 105 (range, 4.6 x 104 to 2.5 x 105) per kilogram of body weight (BW), which corresponded to a mean o~ 1. 2 X 103 (range, 4.6 x 104 to 2.5 x 105) Cl~U-GM/kg BW baced on colony assays. Pelvic aspirates provided the highest density o~ mononuclear cells. Good harvests were difficult to obtain from dogs weighing -10 kg or less because the pelvis t2nded to be too small to allow extensive aspiration; the aspirated marrow was often highly diluted with blood, as indicated by the small proportion of mononuclear cells obtained after density gradient centrifugation. Harvest volumes ranged from 110 mL to 300 mL, representlng 10% to 20% of blood volumes, and transfusion~ ere not required. After culture, the mean number of cells recovered wa~ 1.9 x 107 (range, 1.7 x 1o6 to 4.5 x 107) /kg BW, corresponding to a mean of 8.5 X 103 (range/ 2 . O X 103 to 2.9 x 104~ CFU-GM/kg BW. Thus, on average, the number of cells recovered at the end of culture was 12% of the initial number o~ mononuclear cells seeded, while a smaller proportion o~ the initial n~mber of CFU-GM, about 7%, was recovered a~ter culture.
~FFIC~N~Y O~@~ TR~NæF~R~ RO
Table 2 summarizes data obtained from assays of G~18 resistance, and from detection of theneo gene in on-G-418 resis~ant CFU-~M that wer~ individually plucked (10 to 25 for each dog) and subjected to PCR amplification. In come cases, such as in dog 3, there was a large difference between neo expression, as determined by G418 resistance, and the presence of neo-amplifiable ~ , , ~ . , : , , 2 ~ 4 ~
sequences, as determined by the percentage o~ PCR
positive colonies. The proportion of CFU-G~ resistant to G418 exhibited wide variatio~ about: a mean of 39% and was always le~s than the e~ici ncy of gene transfer measured by PCR. A titration curve or G43.8 re~i~tance is shown in ~ig 2A. We ~ound wide variation in nonspecific toxicity with different lot~ o~ G418. B~cause this was a bioassay, any ~actors that i~libited colony growth would have let to underestimate~ o~'gene 2~pression. The mean gene transfer e~iciency as mea~ured by PCR was approximately ?0%, and ranged ~ro~ 30% in dog 3 to 100%
in do~ 1. The extre~sly high rate of g~ne trans~er a hieved for cultura~ from dog 1 wa3 evident when all CFU-GN colonies plucked for PCR a~pli~ication wera positive for neo (Fig 2B)o Studies of gene transfer e~iciency in cultures after each expo~ure to infection showed that there was a weekly increa~e in the proportion o~ genetically marked cells. This increase was especially evident a~ter the ~inal in~ection (~ig 31. Individually plucked CFU-GM
exhibited a similar increa~e in gen~ transfer ef~ici~ncy and G418 resistanca (data not shown).
TRa~8PL~X~ÆIO~ O~ ICAL~Y M~R~D ~ C~h~8 ~NTO
A~O~O~O~ R~C~P~B~R
~y micro~copic examination, the trypsin~zed cell ~uspen~ion obtained for kransplantation con~isted largely of single mononuclear and spindle shaped cells along with occasional muIticellular clump~ of more than 100 cells.
AdversQ reaction~ to bolus intravenou~ infuYion o~ the cell su~pen~ions ~re not noted. MC+ and control dogs underwent a period of prolonged pancytopenia marked by febrile episocles, leading t~ recovery of cells (neutrophils >l.O x 109/L) by about 36 day~ and platelets (~50 x 109/L) by about ~2 days postirradiation. MC- dogs :
2~8~
did not deYelop any physical or hematologic abnormalities. The procedures per~ormed, dosage~ of cells inje~ted, and duration of follow-up for each dog are outlined in Table 1.
Specimens of peripheral blood obtained from MC+ and MC - dogs at 2 we2ks to 2 1 monthR after infusion o~
cultured cells were consistently positive for neo, as shown by PCR amplification (Fig 4). Similarly, specimens o~ B~ obtained ~ro~ MC+dogs (from 1 month to 21 month after in~usion of cultured cells) and MC - dog~ (from 1 month to 19 months after infusion) were consistently positive ~or neo by PCR amplification (Tabl~ 3 and Fig 5). All five dog~ tested were also positive ~or the neo marker when peripheral blood activated T cell~ were assayed (Table 3). Semi~uantita~lve estimates o~ the retention of neo were made by comparing standardized dilutions o~ the neo amplification product, vi~ualized in gels and Southern blots, with control ~amples o~ 100~, 10%, 1% and 0.1~ dilutions of the cell lines Hh60, N2, or PA317.N2 with the respective parent c811 lines (HL60) and PA317j after similar a~plifications (Fig 5 and Table 3).
In all dogs that received infected cells, the proportion of neo marked cells declined over the first 3 to 4 ~onths postin~usion o~ LTMC cells. The three ~C+ dog~ with follow-up at 21, 17 and 14 months (dog~ 2, 3 and 4) appeared to retain the neo gene in 0.1% to 1% of ~arrow cells and (for do~s 3 and 4) activat~d T cell~.
For dogs 2 through 5, WQ plucked individual CFU-GM, BFUE,-or CFU-Mix colonie~ obtained ~rom marrow aspirates and assayed for th~ presence of neo ~Fig~. The percentag~ of po~3itive colonies generally corrslated with the level of marker retention obtained by visual den~ito~etric estimates based on PC~ amplification o~ DNA
extracted ~rom uncultured marrow cells (Table 3). For example, with dog 2, at 3 months posttran~plant we found 2 ~ 4 ~
~ 27 SL276 that 1 o~ 16 plucked CFU-GM wa~ positive ~or neo, and this compared favourably with the estimated 1% to 10%
positivity based on amplification of DNA from uncultured marrow; at 13 months posttransplant, all 35 CF~-GM
plucked and subjected to PCR a~plification were negative, but analysis of DNA extracted from marrow cells indicated a level of po~itivity corresponding to th~t expected if about 0.1% of c211s in the sample were genetically marked (Fig 5).
There wa~ a seven~old variation in the number of marked cells injected into the variou~ dogs (~able l).
Dog 2, which still retained m~rked marrow cells at 21 month~ received one of the largest doses of genetically marked cells. The data suggest that the proportion of marked marrow cells in ~C~ dog~ decreased from 1~ to 10%
over the fLrst 1 to 4 months but then stabilized at -0.1%
to 1.0% after 3 to 6 months.
Although helper viru~ waR not detected n the PA317- : :
N2 packaging line, it wa~ important to conclusively establish tha~ there wa~ not viru~ spread in vivo.
Pla~ma from the dogs wa~ tested both for the pre~ence of infectiou~ neo viru~ and replication co~petent helper virus. No viru5 was detected in either a~ay (Table 4).
Thus, in contrast to prior art protocol~, successful multiple infections occurred with a retroviral vector of hemotopoietic cells maintained in vitro in LT~Cs.Por thrse weeks, wherein ths infection was timed to coincide with the activation of cell proliferation believed to occur after media replenishment in LTMC . Using a retroviru~ bearing the gene ~or re~istance to the neomycin analog G418 (NQO~ ~, an average in vitro gene transfer e~ficilency greater ~han 70% into LTnC-derived CFU-GM was obta:ined.
. The ulti~ate target of gene transfer into hematopoietic t.Lssue is the pluripotent stem cell, which 2~g$~
can only he indirectly assayed by actually determini~g its potential for hematopoietic reconstitution. There is evidence that such primitive progenitor cells are maintained in LT~C~.I3 To as~ess the efficiency with which the above reporter neo gene may have been transferred into this cell compart.ment, the genetically marked ~TMC cell~ were transplanted into autologous recipients. All dogs were followed-up for 10 to 21 month~ and molecular genetic evid~nce obtained that genetically marked myeloid and lymphoid h~matopoietic cells and their progenitors were present in all five do~c (3 through 7) for which both compartments were examined, suggesting that primitive recon~tituting progenitor cells were indeed infected by thi~ protocol. Infu~ion of less than 104 infected CFU-G~/kg BW r~ ulted in d~tectable engraftm~nt o~ marked marrow progenitors ~or at least ~1 month~. In five dogs, marked marrow cell~ were retained for at least 1 year.
To confirm that the present method provides the acceptable toxicity prer~quisite, ~tandard LT~C~
techniques were modified to maintain prog~nitor cells while achieving highly efficient infaction.
Infective retrovirus wa~ prepared in LTMC media in an attempt to condition the madia and further enhance culture viability. Omi~sion of cocultivation steps p.evented any potential 1058 0~ pluripotent stem cells due to adherence33 to virus producing fibroblasts.
Omission of toxic selection by exposure to G418 permitted establishment of LT~C with simple -protocols u~compli~ated by exhaustive marrow harvasts, growth factor~, or f~eder layers.
The role oiE marrow-ablative conditioning represents a third signi~icant consideration in the clinical implementation o~ gene therapy addressed in this specification. The re ults of previous studies suggest 208~
- 29 ~ 5L276 that the dosages of irradiation herein used ~or marrow conditioning were high enough to be lsthal in the ab ence of rescue by transplantationO~43~ In this specification, however, dogs undergoing marrow conditioning received vigorous support including da.Lly transfusion of irradiated blood products and comprehen~ive a~tibiotic coverage when necessary.39 Thil~ level of 3upport permitted th~ hematopoietic recovery of dog~ 8 and ~ -a~ter they received the conditioning regimen but no infusion of LTMC cells. For dog~ that received a full transplant protocol, it is con¢luded that while some of the transplanted cells undoubtedly engrafted and contributed to recovery, thQ LTMC cells were not essential for recovery. In this context, it was surprising that a ~ignificant number of genetically marked cells in vivo were detected compared with th~ work of others.~7~ Thes~ result-~ indicat~ that marrow progenitors maintained and retrovirally infected in L~MCs can engra~t in the face of a presumably ~trong endogenous recovery of marrow during the pancytopenic postirradiation phase.
2Q~8~'~
_ 30 - 8~276 T5bb a ~oa d P~ ~ h ~ eUb Al~ ~ T~l~ cO LT~g Cdb T _le~ M~ a~ _ w ~ w 1~10 N~ 1~10 ND 1 3 1.10 l/tll 1 Nl:l 0.1~1 V10 O.t~ 0 0.1-1 0 0.1~1 NO Ø1-1 9/30 Q1.1 Nl~
7 0.1~1 1/3D
0.014.1 31~a 0.1.1 12 0.1~ V~7 ~ 1 0.1-1 3/10 0.~1 14 0.1-1 0131~
1~1 Q~-t 019 7 0.1~1 Vl~ 0.1~1 ~1 Q1~1 3/11 7ll~ d ~ T ~ ~ h ma _ q ~ Al~_ d r~ 9#p~ h ON/~ o~l bam 1~ to al o o 3 1~ 0 0 12 0 1:1 1~ 0 0 ~I 12 0 0 PA31t~2 19' N~o N O
_ _ m~
2 ~
Figures 7a, 7b and 7c show that tha levels of engra~tment observed equalled or surpassed thoss obtained previously when dogs were sub~ecte.d to marrow ablative therapy prior to the infusion of miarked cells.
Th~ present results indicate that; (1) LTMC cells can reconstitute the hematopoietic systems of large animals; ~2) Marrow ablative conditioning is not necessary ~or engraftment of LTMC cells and may, in fact, cQmpromise engraft~ent by upregulating endogenous hematopoiesis; (3) Only a few stem cells are cycling at any given time in large mammals; an~ (4) I~L vitro activated st~m cells complete normal differentiation and proliferation programs when returned to the ~nL_YiYQ
microenvironments ~rom whence they came.
While the inv~ntion has been dèscribed in detail and with reference to specific embodiments thereo~, it will be apparent to one skilled in the art that various changes and modifications can be mada therein without deporting from the spirit and scope o~ the invention.
.
29. Ekhterae D, Crumbleholme T, Karson E, Harrison MR, Anderson WF, Zanjani ED Retroviral vector-mediated transfer o~ the bacterial neo~ycin resistance gene into fetal and adult sheep and human hematopoietic progenitors in vitro. Blood 75:365, 1990.
30. Bodine DM, Karls~on S, Nienhui~ AW Co~bination of interleukins 3 and 5 preserv2s st m cell function in culture and ~nhances retrovirus-medi~ted gene transfer into he~atopoietic ste~ cells. Proc Natl Acad Sci USA 86:8897~ 19~9.
lS 31. Fletcher F, Williams D, Mali~w~ki C, Anderson D, Rives M, Belmont J: ~urine leuX mia inhibitory factor enhances r~troviral-vector infection efficiency of hematopoietic progenitors. Blood 7Ç 11~98, l990o 32. Dick JE, Kamel Reid S, Murdoch B, Doeden~ M: Gene transfer into normal human hematopoietic cells u ing in vitro and in vivo as~ays. Blood 78:624, 1991.
33. Gordon ~Y Adhe~ive properties o~ haemopoietic stem cells. Br J Haematol 68:149, 1988.
34. Toksoez D, Dexter TM, Lord BI, Wright EG, Lajtha LG:
The regulation of hemopoe~is in long-term bon~
marrow culture II. Stimulation and inhibition of stem cell proliferat~on. Blood 44:931, 1980.
35. Cashman J, Eaves AC, Eaves CJ: Regulated proliferation of primitive hematopoietic progenitor cell~ in long-term human marrow cultures. Blood 66:1002, 1985.
36. Carter RF, Kruth SA, Valli VE0, Dub~ ID: Long-term culture of canine marrow: Cytogenetic evaluation of purging of lymphoma and leukemia. Exp. Hematol -'` 2~8~
- g SL276 18:995, 1990.
37. Armentano ~, Yu 5, Kantoff PW, von Ruden T, Anderson WF, Gilboa E: Effect of internal virus sequences on the utility o~ retroviral vectors. J Virol 61:1647, 1987.
38. Saiki RK, Scharf St Faloona F, Mulli~ KB, Horn GT, Erlich H~, Arnheim N: Enzy~atic amplifi~ation of B~
globin genomic sequences and restriction ~ite analysis for diagnosis of sickle cell anemia.
Science 230:1350t 1985.
39. Abrams-Ogg ACG, Kruth SA, Carter RF, Valli VEO, DUba ID, Dick JE: Autologou~ bone marrow transplantation u~ing long term bone marrow culture in dog~. J Vet Intern Med 4~130, 1990 (abstr).
40. Storb R, Raf~ RF, Appelbaum FR, Graham TC, Schuening FG, Sale G, Pepe M- Compari~on of ~ractionated to single-dose total body irradiation in conditioning canine littermates for DL~identifical marrow grafts. Blood 74:1139, 1989.
41. Kalebo M. Garcia JV, Osborne WA, ~iller AD:
Expression of hu~an adeno~ine dea~inase in mice after tran~plantation of genetically modified bone marrow. ~lood 75:1733, 1990.
42. Joyner A, Keller G, Phillip~ R~, Bern3tein A:
Retroviru~ transfer o~ a bacterial gene into mouse hematopoietic progenitor cells. Natur~ 305:556, 19~3.
43. Storb R, Raff R~, .Appelbau~ FR, Schuening ~G, San~maier B~, Graham TC, Thomas ED What radiation c~o~e ~or DL~-identifical canine marrow grafts?
Blood 72:1300, 1988; erratum Blood 73:624, 1989.
44. Down JD., Tarbell NJ, Thames HD, Mau~h P~: Syngeneic an~ allogeneic bone marrow engraftment a~ter total body irradiatio~: Dependencs on dose, does rate, and fractionation. Blood 77:661, 1991.
2 ~
.
45. Mic~lem HS, Ford CE, Evan~ EP, Ogden DA: :
Compartmen~s and cell ~low~ within the mouse haemopoietic system. I. Re.stricted interchange between haemopoietic site~. Cell Tissue Kinet 8:219, 1~75.
46. Micklem HS, Ogden DA, Evans EP, Ford CE, ~ray JG:
Compartments and cell flow~s within the mouse haemopoietic system. II. Estimated rates of interchange. C~ll Tissue Kinlet 8:233, 1975.
~, ~U~ Y 0~ T~ INV~N~IO~
It is a~ object of the present invention to provide an improved protocol for gen transfer into receptive targPt cells, target cells for use in enhancement of the genetic material of living organi~ms, such a~
microoryanisms, ~ungi, pl2nts and animals, including stem cells and target c~118 for transient expression, It is a further object of the present invention to provide desired gene-containing target cells and compo~ition~ of u5e in medica~ent~ ~or ma~al~ and methods o~ treatmen~ o~ the ~mmal3 using ~aid cells.
It is a yet further object of the invention to provide a method ~or activating stem cells and a method for the adoptiv~ tran~fer o~ ~aid cells.
Thu~; the method according to the invention compri6es subjecting cells in long term culture to repeated exposure to ~ene transfer vahicle~, particularly retroviru~ vector, preferably ti~ed to coincide with the wave o~ cellular proliferation thought to occur a~ter media replanishment. While not being bound by theory, it is bslieved that the method accordlng to the invention allows stem cel:Ls maintained in culture to be triggered -' 2~8~
into activated cell-cyle status thereby providing the opportunity for optimizing retroviral infection over a relatively long period of time while simultaneously creating a population of activated stem cells with the potential ~or extensive proliferat.ion n vivo.
Accordingly, in its broadest a~pect the invention provides a method o~ effecting trans~er of a gene into a gene tran~fer target cell comprising ~tabli~hing a long ter~ culture containing said cell~ in a uitable medium and exposing said culture to multiple, periodic infection~ of a gene transfer vehicle containing said gene to provide said gene - containing target cell.
Preferably, the media is periodically, partially substituted with fresh media.
15 The vector is provided in an amount effective to transfer the gene into the cells, and provided at the steps o~ development o~ the cells such that the cell~ are capable of being in~ected by the vector and infusion of marked ~te~ cells without conditioning host.
While of general applicability for ef~icient gene trans~er in receptive target cell sys~em~, the met~od of the in~ention is of particular value in the production of long term marrow culture cells genetically marked by retroviral vectors and infusion of marked ~tem cells without conditioning of host.
The methods according to the invention are applicable to all receptive target cell~, particularly stem cells such as epithelial stem cells, exocrine gland stem cells, endocrine gland stem cells, stromal stem cells,- long term culture initiating cells,-hepatocytQ
stem cells, pancreatic stem cells, neuroendocrine stem cells, connective tissue ~tem cells, ~ibroblast stem cells, mesench~nal stem cells, adipose cell tem cells, mammary gland stem cells, reticuloendothelial stem cells, lipid ste~ cells, chondrocyte stem cells, oste4progeni~0r 2 0 ~
- 12 ~ SL276 stem cells, osteocyte stem cells, muscle fiber stem cells, neuronal stem cells, epidermal stem cells, keratinocyte stem cells, Langerhans stem cells, m~lanocyte stem cells, sebaceous gland ste~ cells, sweat gland stem cells, mucous stem cells, serous cell stem :
cells, odontoblast stem cells, islet of Langerhans stem cells, alveolar stem cells and ret:inal stem cells.
In addition, the invention i~ of value in providing modifications to target cells with less cap~city ~o~ cell renewal by modifying the duration of culture, the retroviral titer, the retrovirus, timing and durakion of exposure to gene transfer vehicle, and other parameter3, it is possible to specifically target cell~ that have specific capacities for proliferation. Thi~ approach will have applications in gene ~therapy for somatic disease where transient expres~ion of the gene is a desirable outcome. Target cell~ for trancient expression include mast cell~, eosinophils, plasma cells, lymphocyte~, monoblast~, promonocyte~, monocytes, macrophages, basophils neutrophil~, erythrocytes, granulocytes, megakaryocytes, platelets, osteoblasts, osteocyte~, osteocla~ts, ~ibroblast~, epithelial cells, stromal cells, hepatocytes, pancreatic cells, neuroendocrine cell~, connective ti~sue c 11R, mesenchy~al cell~, adipocytes, mammary gland cells, reticuloendothelial cells, lipid cells, chondrocytes, muscle cells, neuron~, epidermal cells, ~angerhans cells~
melanocytes, gland cells,. mucous cells, serous cells, odontobla~ts, i~let of Langerhans cells, alveolar cells and re~inal cells.
Pref erably, the method as hereinabove de~ined comprises establishing said culture in a first suitable medium; exposinl~ said culture to a first infection of said gene transfer vehicle; including said infected cultùre for a sufficient period o~ time to ef~ect cell 2~8~44 proliferation; substituting a portion of said firs~
suitable medium with a second ~uitable medium to provid2 a first resultant medium; exposing said culture to a second infection of said gene transfer vehicle; further incubating said inf~cted culture for a sufficient period of time to e~fect ~urthe~ cell proliferation and in vitro cell renewal; and recovering said gene-containing target cell as w~ll.
More preferably, the method f~lrther co~pri es prior to recovering said gene-containing target cell, substituting a portion of said ~irst resultant mediu~
with a third suitable medium to provide a ~econd resultant medium; exposing said culture to a third infection of said gene transfer vehicle; and further incubating said culture for a sufficient period of time to effect yet further cell prolif~ration and in vitro cell renewal.
The gene-trans~er vehicle may be added simultaneously to the cultures with the exchanging ~edia, but, preferably, each mediu~ i~ added a few hour~ be~ore, pre~erably, say, 10 hours bs~ore or more preferably the day before the gene-transfer vehicle is added to the resultant culture.
The cultures are ~llowed to incubate ~or sufficient periods of time to effect cell pxoliferation, typically at least ~or 3 days but preferably at least 7 days.
These significant period~ of time for incubation in an environment that ~imulates in vivo microenvironment allow~the stem cells to proliferate and di~ferentiate by activa~ion and lenhancement o~ the ~elf-renewal ~yste~.
Multiple, partial renewal of the media, periodically, encourages the progenitor cells to address ~he shock to the cell system by 3timulating activation o~ the entire progenitor cell hierarchy available for qene-transfer.
~S8~
It is widely beli~ved that in vivo, microenvironment~ that normally support the proliferation and differentiation of stem cells must be appropriat~ly conditioned if they are to receive and support 'the proliferation of exogenously administered stem cells.
Thus, many investigators elect to subject the recipients o~ genetically manipulated hematopo:ietic cells to marrow ablative conditioning prior to adoptiv~ gene tran~ferO
The assumption is that the he~atopoietic cells provided post-marrow ablation will home to the "empty'i marrow microenvironment, theoretically ideally suited for their maintenanc~.
Thexe is evidenc~, however, that in large mammals the maximum death-sparing level of irradiation ic not totally marro~ablative. If animal~ subjected to such irradiation are vigorou~ly supported during the pancytopenic post irradiation phase, their hematopoietic system~ eventually recover. During this recovery phase, the animals' hematopoietic systems are most likely subjected to self-regulatory feedbacX control mechanisms that strongly support the proliferatio~ of thos~ stem cells would presumably face strong co~petition from their endogenou~ counterparts.
We hav~ di~coversd that in vivo m~intenance of ~B
yitro activated hematopoietic ~tem c~113 have been signi~icantly enhanced in recipient3 that wera not subjected to a~y type o~ marrow conditioning. LTMCs fro~
15 dog3 were established and subjected to gene transfer using 21-day, 3 cycle transduction protocol with th~ N2 -30 ratroviral vector. The dogs were divided in~o 3 groups of 5 each. ~ach group received a different number of infu~ed LTMC cells. No dogs underwent marrow conditioning. Dog~ were followed a~ before for evaluation of the contribution mad~ to hematopoie~i~ by the LTMC cQll~.
8 ~ ~
- l5 - SL276 Accordingly, in a further aspect the inven ion provides a method of enhancing 1:he proliferation o~
pluripotent stem cells in vivo in a mammal comprising administering activated stem cells a~ hereinabove defined to said mammal and allowing proli~eration of said c~
in said mammal in the absence of ablative marrow conditioning.
By the term "ablative marrow conditioning" is meant the removal of all host cells from a particular site in thq recipient, which c~lls include abnormal and nor~al tissue. Such abnormal cells ganerally comprise gen~tically defectiv~, genetically deficient, mutated, cancerous and virally in~ected cells. Ablative marrow conditioning include~ chemotherapy and irradiation treatments.
In yet a further a~pectV the invention provides a method ~or activating quiescent repopulating stem cells in vitro to provide activated stem cells which maintain their activity in vivo comprising establishing a long term culture containing said quiescent ~tem cell~ in a suitable m~dium and m~ltiple, periodic partial substitution o~ s~id medium to provide said activated stem cell and recovering said activated ste~ cell.
PreferablyO the method comprise~ establishing a method as h~reinabove defined compri ing es~ablishing said culture in a ~irst suitable medium; incubating said culture for a first su~ficient period of time to ef~ect cell proliferation and. ~ vitro cell renewal;
substituting a portion of said fir~t suitable medium with a second suitable medium to provide a ~irs~ resultant medium; further incubating ~aid culture for a second sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said actlvated stem Cell7 ':
: ' ~ ' . ' ' .
'' ''" ' ~
2~8~84~
The pre~erred periods of time to e~fect cell proliferation is at least 3 days.
D I~D ~8CRIP~I0~ OP ~ IN~I0 In order that the invention may be better under~tood a preferred embodiment will now be described, by way of example only, with reference to the accompanying drawings, wherein:
Figure 1 represents a bar chart summarizi~g the numbers of cells and CFU-GM harvested ~rom nine dog~;
Figure 2 represents ~A) a titration curve ~or G418 resistance; and (B) agarose gel electrophores~3 of neo~
a~plified sequence~ from dog 1;
Figure 3 represents southern analysis o~ neo-amplified sequences in LT~C adherent layer cells from dog 3;
Figure 4 repre~ent~ agarose gel electrophoresiR o~ ~eo-ampli~ied sequences in peripheral blood cells obtained from dogs 2, 3 and 5 a~ter transplantation;
Figura 5 represents southern analysis neo-amplified seguence~ in marrow aspirated from dog 2 at 13 months posttransplantation; and Figure 6 repre~ents agarose gel electrophone~i~ of neo-amplified sequenceso individually plucked colonies obtained fro~ dog 3 at 8 month~ posttransplant.
Figure 7 represent~, as Figure 7a, Figure 7b and Figure 7c percentages of .G418 resistant marrow-derived hematQpoietic progenitors for several group~ of dogs.
~ong term marrow culture from normal dogs w~s exposed to ~hree round~ o~ infection with neo~containing retrovirus produced by the packaging cell line PA317-N2 during twenty-ona day~ of culture. The results showed that the nQomycin phosphotransferase gene (neo) wa~
transferred with high efficiency into hema~opoietic ~ o ~
progenitors in LTMCs. After infusion of infect~d LTMC
cells into autologous recipients, were detected progency cells of genetically marked marrow progenitors, with and without the use of marrow condit-ioning by total body S irradiation ~TBI~.
~A~R~A~ A~D ~T~OD8 Normal dogs were obtained ~rom the Central An~mal Facility of the University of Guelph and maintained in the Veterinary Teaching Hospital until they recovered from all procedures. All protocols were approved by committee~ of the University of Toronto. The Toronto Hospital f and the University of Guelph concerned with animal care, biohazards, and ethical review.
p~pARa~rIo~ o~ VI~ $QP13R~ 8 The necl retroviral vector used wa. the Moloney murlne leukemia-derived vector, N2,~ 8e~ore ~arrow harvest~ the help~r cell line PA317.N2 was grown in a minimal essential medium (alpha-Me~) media containing 4.5 g/L l-glutamine, 10% fetal bovine serum, and 0.2g/L G418.
The day before marrow harvest, the alpha-~EM media was replaced by media used ~or LTMCs . 36 On the day of the marrow harvest, this media was collected and a cell free supèrnatant co~taining -1 x 106 viruslmL ln long-term culture media was prepared by centrifugation at 400g for 15 minutes. Ali~uot~ o~ viru~-containing media were similarly prepared for second and third infection~ of each culture. No helper viru~ was detected by a sensitive viral rescue assay in the~e viral ~upernatants or from th~ ~T~C~ after 21 day . .
2Q8~
GEN~ TRaN~FBR INTO C~NINB ~C
After induction o~ general anesthesia, marrow and blood cells were aspirated from the iliac crests, ischiatic crests, and proximal femori and humeri o~ each dog, and placed directly into preservative ~ree heparin (2,000 U/lOOmL of aspirated marrow). After determination o~ the total viable nucleated cell count ~y trypan blue exclusion, the mononuclear cell fras:tion was prepared by density centri~ugation and LT~Cs were initiated as prepared by density centrifugation and LTMCs were initiated as previously described 36 along with the following modifications: each marrow culture was set up in culture m~dium containing retrovirus and exposed to virus on two more occasion~ during the culture period, with the last exposure to virus occurriny 6 day~ be~ore culture harvest and in~usion at 21 days.
Specifically, cultures were initiated by inoculatillg Corning T-150 ~lasks (Corning, Corning, NY) with 1~2 x 1o8 mono~uclear cells in 60 mL of media containing virus.
2 0 On days 7 and 14 of incubation, thQ f lask wQre demi-depopulated and fed with non-virus containing media. On days 8 and 15, on half of the supernatant layer of each flask was removed, the cells were pe.lleted and resuspended in fresh media containing ~1 x 106 virus/mL.
A~ter 21 day3 of culture, all adherent cells were recovered by trypsinization, washed three times in presexvative free, pH indicator free Hank's buffered saline solution (GIBCO, Grand I~land, NY), and kept at room temperatur~ for 1 to 3 hours before bolu -injection b~ syringe and butterfly needle infusion set into the cephalic vein of the autologous recipient.
. , . . . ~ .~ . ,;
2~84~
PRo~ITO~ A88~Y~
Progenitor cells (colony ~o~ming unit granulocyte macrophage [CFU-GM], burst fvrming unit erythroid [BFU-E], and CFU-~ix) were a~sayed, at least in duplicate, in S 1 mh of methylcellulose media plated in 35 mm petri dishes as previously described.36 Resistance to the ~eomycin analog, G418, was assess;ed by counting the numbers of colonies ari~ing by day 10 in plates containing 1 mg/m$ G418 comp~red with plates containing no added G418. For evaluation o~ T cells, nucleated peripheral blood cells were maintained in liguid culture consisting RPMI 1640 IGIBC0), 10% fetal calf serum (FCS), 1% L glutamine, and 1~ pen/strep, at 106 cells /mL.
Phytohemagglutinin (PMA; Wellcome, Dartford, U.K.) was added for 72 hours at culture initiation after which time cells were pelleted by centri~ugation and resuspended in fresh media supplemented with interleukin 2 (Cetus, Norwalk, CT) at 20 U/m~ twice weekly ~or 2 to 3 w~eks.
DBT~C~Io~ o~ o ~Y PO~ 8~ c~aI~ REac~ C~) :
Approxi~ately 106 cells fro~ ~resh blood, BM, cultured T c~ , or LTMCs were lysed and DNA was extracted ~or PCR ampliPication. DNA from day 10 CFU-GM, growing in the absence o~ ~418 and estimated to contain at least 20 cells, was obtained for PCR amplification by placing plucked colonie~ dir~ctly in 19 ~ of extraction buffer (50 m~ol/L XC1, 10 mmol/L Tri~ HCl pH 8.3, 2.5 mmol/L ~GC12, 0.1 mg/mL 2~ gelatin, 0.45% nonidet P40, O.45% ~ween 20~ and incubating with 10 proteinase K for 2 hours at 55C. The neo-specifi¢ oligonucleotide primeers were a 21 mer to the sen~s strand (5' AAAG~ATCCATCATGG~CTGAT 3'), and a 20mer to the antisense strand (5' ATGt'TCTTCGTCCAGATCAT 3'). Neo specific sequence~ were amplified according to standard CPtus protocol38 from 250 ng to ~g extracted DNA or DN~ obtained 2 ~ 4 ~
by direct lysi~ oP plucked colonies. The denaturatlon temperature wz3 94C, the annealiny teMp~rature was 68C, and the extension temperature waR 72Co ~he amplified DNA was identifi~d a~ter electrophoresi~ in 1% agarose and 2~ NuSi~ve G~ agarOsQ (FMC b.ioproducts, Rockland, ME) by staining with ethidium bro~ide. Southern blot~
were prepared by transferring the DNA fro~ the gel to a nylon membrane (Hybond; Amersham, Oakville, ontario, Canada). Blot3 were probed with a 32p labelled neo fragment. Specificity wa~ deter~irled by autoradiography with Kodak X-omat ~ilm (Rochester, NY~ Control specimens included serial dilutions of PA317.N2 and/or HL60N2 (an HL60 cell line containing a neo vector), reagents snly, BM cells from uninfected nor~al dogs, and CFU G~ derived ~ro~ as3ays o~ uni~fect~d normal canine marrow. Samples were con~idered positiv~ i~ a band identical to the positive control wa detectabl~ by ethidium bromide staining of the gel. Southern blots were used to confirm that suspected positives actually were neo specific se~uences.
MARRO~ ÇO~DI~IO~ A~D ~a~RO~ TRa~A~A~IO~
Dogs received identical preparative and isolation protocol ; details o~ the prQparativ~, irradiation, and support protocols have been described elsewhere.39 Marrow conditioning wa~ administered to transplant dogs and control dogs by bilateral ex~osure to a single ~Co source; the total midline dose was 6 Gy or S Gy in a single- ~raction at dose rates fro~ 10 cGy/~in to 75 CGY/~rn. DOgR recei~ing transplants without marrow conditioning wer~ not irradiatad and wer~ not administered antibiotics. Follow up speci~ens of periphèral blood and BM (obkained under general anesthesia) wer~ obtained at intervals and assayed as 2 ~
.
- 21 ~ SL276 described above by PCR amplification for the presence o~
neo.
D~Æ~IO~ O~ I~F~C~O~ O YI~ AND ~ CA~ION
CONP~ R~TR~VI~8 I~ PhA8~A OR R~CO~ BD DO~8 The PA317-N2 cell line was re~established from frozen stocks every 6 weeks to pre!vent the inadvertent production of replication competent virus. Plasma ~rom reconstituted dogs wa tested for thQ presence of infectious neo viru~ using Rat 2 cell~ a~ an in~icator line. Dog plasma was diluted to 10~ with a-MEM
containing 8 ug/mL polybrene and incubated ~or 24 hours with 2 x 105 Rat 2 cells on 60 mm tissue culture plates~
Higher plasma concentration~ cau~ed Rat 2 cells to grow poorly. After 24 hours, ~resh a ME~ wit~ 400 ug/mL G418 was added to the plates. Th~ plates were incubated ~or 7 days a~d scored for ~418R colonie~. The positive control was the PA317-Na ce~l line.
The dog plasma was also tested for the presence of replication competent helper virus using a marker resuce assay.4~ The diluted plasma, alpha-ME~, and polybrene were incubated with NeoNP-l cells ~or 24 hour~. N~oNP-l is a 3T3 cell linQ in~ectQd with ths MLVNEO.1 retrovirus vecto~2. Any helper viru~ in the test sampl~ will spread throughout the culture and resuce the ~LVN~O.1 vector that can then b~ assayed on Rat 2 cell~ or human HOC-7 cell~. The NeoNP-1 cells were passaged and grown for at least 1 week to permit viru~ spread. This supernatant wa~ diluted fro~ 10 to 10-3 and incubated with Rat 2 cells-to as~ay for the rescued MLVNEO.l vector. The positive control was supernatan~ from a packaging cell line known to ma:ke helper virus and the negative control was supernatant fro~ the NeoNP-1 cell line.
2 ~
RE8U~T8 - Tables 1 and 2 summarize the procedures and results for nine dogi~. Four dogs received the entir~ transplant protocol including marrow condi.tloning ("~C~", dogi3 1 through 4). The first dog di~d due to a failure of support (~ulminant Streptococcal septicemia) at 18 dayis postirradiation, but dog~ 2, 3 and 4 survi~ed and were . evaluated for evidence of retenti.on of genet~cally ~arked ;. hematopoietic celli3. Thre~ dogs rQceived the entire protocol except that marrow conditioning was completely omitted ("MC-", dogs 5, 6, and ?). Dog3 ~ and 9 were controls that received the entire protocol except that no cells of any kind were infused after marrow conditioning;
others recovered ~rom pancytopenia at approximately 35 days but dog 9 died after a sever~- acute rQaction to a platelet transfusion at 36 days.
:.,:, . . ~ . ... . , - . , r~ 2 ~ 8 ~ 4 J . ~
i a ~ a21!~
i ~ ~x I ~ x ~ J l ~ i o ~ ~ ~ E
i~ 3J~ ~t ~ i~
¦~¦ Jll~
3 ~ ~
24 - SL~76 NARRO~ ~A~V~8 AND_C~ R~COV~Y FRO~ C~L~ 8 Figure 1 summarizes the numbers of cells harvested and then obtained after 21 days of LTMC. The mean number of mononuclear cells initially harvested was 1.6 x lo8 (range, 3.1 x 107 to 2.3 x 108) per kilogram of body weight (BW), which corresponded to a mean o~ 1.2 x 105 (range, 4.6 x 104 to 2.5 x 105) per kilogram of body weight (BW), which corresponded to a mean o~ 1. 2 X 103 (range, 4.6 x 104 to 2.5 x 105) Cl~U-GM/kg BW baced on colony assays. Pelvic aspirates provided the highest density o~ mononuclear cells. Good harvests were difficult to obtain from dogs weighing -10 kg or less because the pelvis t2nded to be too small to allow extensive aspiration; the aspirated marrow was often highly diluted with blood, as indicated by the small proportion of mononuclear cells obtained after density gradient centrifugation. Harvest volumes ranged from 110 mL to 300 mL, representlng 10% to 20% of blood volumes, and transfusion~ ere not required. After culture, the mean number of cells recovered wa~ 1.9 x 107 (range, 1.7 x 1o6 to 4.5 x 107) /kg BW, corresponding to a mean of 8.5 X 103 (range/ 2 . O X 103 to 2.9 x 104~ CFU-GM/kg BW. Thus, on average, the number of cells recovered at the end of culture was 12% of the initial number o~ mononuclear cells seeded, while a smaller proportion o~ the initial n~mber of CFU-GM, about 7%, was recovered a~ter culture.
~FFIC~N~Y O~@~ TR~NæF~R~ RO
Table 2 summarizes data obtained from assays of G~18 resistance, and from detection of theneo gene in on-G-418 resis~ant CFU-~M that wer~ individually plucked (10 to 25 for each dog) and subjected to PCR amplification. In come cases, such as in dog 3, there was a large difference between neo expression, as determined by G418 resistance, and the presence of neo-amplifiable ~ , , ~ . , : , , 2 ~ 4 ~
sequences, as determined by the percentage o~ PCR
positive colonies. The proportion of CFU-G~ resistant to G418 exhibited wide variatio~ about: a mean of 39% and was always le~s than the e~ici ncy of gene transfer measured by PCR. A titration curve or G43.8 re~i~tance is shown in ~ig 2A. We ~ound wide variation in nonspecific toxicity with different lot~ o~ G418. B~cause this was a bioassay, any ~actors that i~libited colony growth would have let to underestimate~ o~'gene 2~pression. The mean gene transfer e~iciency as mea~ured by PCR was approximately ?0%, and ranged ~ro~ 30% in dog 3 to 100%
in do~ 1. The extre~sly high rate of g~ne trans~er a hieved for cultura~ from dog 1 wa3 evident when all CFU-GN colonies plucked for PCR a~pli~ication wera positive for neo (Fig 2B)o Studies of gene transfer e~iciency in cultures after each expo~ure to infection showed that there was a weekly increa~e in the proportion o~ genetically marked cells. This increase was especially evident a~ter the ~inal in~ection (~ig 31. Individually plucked CFU-GM
exhibited a similar increa~e in gen~ transfer ef~ici~ncy and G418 resistanca (data not shown).
TRa~8PL~X~ÆIO~ O~ ICAL~Y M~R~D ~ C~h~8 ~NTO
A~O~O~O~ R~C~P~B~R
~y micro~copic examination, the trypsin~zed cell ~uspen~ion obtained for kransplantation con~isted largely of single mononuclear and spindle shaped cells along with occasional muIticellular clump~ of more than 100 cells.
AdversQ reaction~ to bolus intravenou~ infuYion o~ the cell su~pen~ions ~re not noted. MC+ and control dogs underwent a period of prolonged pancytopenia marked by febrile episocles, leading t~ recovery of cells (neutrophils >l.O x 109/L) by about 36 day~ and platelets (~50 x 109/L) by about ~2 days postirradiation. MC- dogs :
2~8~
did not deYelop any physical or hematologic abnormalities. The procedures per~ormed, dosage~ of cells inje~ted, and duration of follow-up for each dog are outlined in Table 1.
Specimens of peripheral blood obtained from MC+ and MC - dogs at 2 we2ks to 2 1 monthR after infusion o~
cultured cells were consistently positive for neo, as shown by PCR amplification (Fig 4). Similarly, specimens o~ B~ obtained ~ro~ MC+dogs (from 1 month to 21 month after in~usion of cultured cells) and MC - dog~ (from 1 month to 19 months after infusion) were consistently positive ~or neo by PCR amplification (Tabl~ 3 and Fig 5). All five dog~ tested were also positive ~or the neo marker when peripheral blood activated T cell~ were assayed (Table 3). Semi~uantita~lve estimates o~ the retention of neo were made by comparing standardized dilutions o~ the neo amplification product, vi~ualized in gels and Southern blots, with control ~amples o~ 100~, 10%, 1% and 0.1~ dilutions of the cell lines Hh60, N2, or PA317.N2 with the respective parent c811 lines (HL60) and PA317j after similar a~plifications (Fig 5 and Table 3).
In all dogs that received infected cells, the proportion of neo marked cells declined over the first 3 to 4 ~onths postin~usion o~ LTMC cells. The three ~C+ dog~ with follow-up at 21, 17 and 14 months (dog~ 2, 3 and 4) appeared to retain the neo gene in 0.1% to 1% of ~arrow cells and (for do~s 3 and 4) activat~d T cell~.
For dogs 2 through 5, WQ plucked individual CFU-GM, BFUE,-or CFU-Mix colonie~ obtained ~rom marrow aspirates and assayed for th~ presence of neo ~Fig~. The percentag~ of po~3itive colonies generally corrslated with the level of marker retention obtained by visual den~ito~etric estimates based on PC~ amplification o~ DNA
extracted ~rom uncultured marrow cells (Table 3). For example, with dog 2, at 3 months posttran~plant we found 2 ~ 4 ~
~ 27 SL276 that 1 o~ 16 plucked CFU-GM wa~ positive ~or neo, and this compared favourably with the estimated 1% to 10%
positivity based on amplification of DNA from uncultured marrow; at 13 months posttransplant, all 35 CF~-GM
plucked and subjected to PCR a~plification were negative, but analysis of DNA extracted from marrow cells indicated a level of po~itivity corresponding to th~t expected if about 0.1% of c211s in the sample were genetically marked (Fig 5).
There wa~ a seven~old variation in the number of marked cells injected into the variou~ dogs (~able l).
Dog 2, which still retained m~rked marrow cells at 21 month~ received one of the largest doses of genetically marked cells. The data suggest that the proportion of marked marrow cells in ~C~ dog~ decreased from 1~ to 10%
over the fLrst 1 to 4 months but then stabilized at -0.1%
to 1.0% after 3 to 6 months.
Although helper viru~ waR not detected n the PA317- : :
N2 packaging line, it wa~ important to conclusively establish tha~ there wa~ not viru~ spread in vivo.
Pla~ma from the dogs wa~ tested both for the pre~ence of infectiou~ neo viru~ and replication co~petent helper virus. No viru5 was detected in either a~ay (Table 4).
Thus, in contrast to prior art protocol~, successful multiple infections occurred with a retroviral vector of hemotopoietic cells maintained in vitro in LT~Cs.Por thrse weeks, wherein ths infection was timed to coincide with the activation of cell proliferation believed to occur after media replenishment in LTMC . Using a retroviru~ bearing the gene ~or re~istance to the neomycin analog G418 (NQO~ ~, an average in vitro gene transfer e~ficilency greater ~han 70% into LTnC-derived CFU-GM was obta:ined.
. The ulti~ate target of gene transfer into hematopoietic t.Lssue is the pluripotent stem cell, which 2~g$~
can only he indirectly assayed by actually determini~g its potential for hematopoietic reconstitution. There is evidence that such primitive progenitor cells are maintained in LT~C~.I3 To as~ess the efficiency with which the above reporter neo gene may have been transferred into this cell compart.ment, the genetically marked ~TMC cell~ were transplanted into autologous recipients. All dogs were followed-up for 10 to 21 month~ and molecular genetic evid~nce obtained that genetically marked myeloid and lymphoid h~matopoietic cells and their progenitors were present in all five do~c (3 through 7) for which both compartments were examined, suggesting that primitive recon~tituting progenitor cells were indeed infected by thi~ protocol. Infu~ion of less than 104 infected CFU-G~/kg BW r~ ulted in d~tectable engraftm~nt o~ marked marrow progenitors ~or at least ~1 month~. In five dogs, marked marrow cell~ were retained for at least 1 year.
To confirm that the present method provides the acceptable toxicity prer~quisite, ~tandard LT~C~
techniques were modified to maintain prog~nitor cells while achieving highly efficient infaction.
Infective retrovirus wa~ prepared in LTMC media in an attempt to condition the madia and further enhance culture viability. Omi~sion of cocultivation steps p.evented any potential 1058 0~ pluripotent stem cells due to adherence33 to virus producing fibroblasts.
Omission of toxic selection by exposure to G418 permitted establishment of LT~C with simple -protocols u~compli~ated by exhaustive marrow harvasts, growth factor~, or f~eder layers.
The role oiE marrow-ablative conditioning represents a third signi~icant consideration in the clinical implementation o~ gene therapy addressed in this specification. The re ults of previous studies suggest 208~
- 29 ~ 5L276 that the dosages of irradiation herein used ~or marrow conditioning were high enough to be lsthal in the ab ence of rescue by transplantationO~43~ In this specification, however, dogs undergoing marrow conditioning received vigorous support including da.Lly transfusion of irradiated blood products and comprehen~ive a~tibiotic coverage when necessary.39 Thil~ level of 3upport permitted th~ hematopoietic recovery of dog~ 8 and ~ -a~ter they received the conditioning regimen but no infusion of LTMC cells. For dog~ that received a full transplant protocol, it is con¢luded that while some of the transplanted cells undoubtedly engrafted and contributed to recovery, thQ LTMC cells were not essential for recovery. In this context, it was surprising that a ~ignificant number of genetically marked cells in vivo were detected compared with th~ work of others.~7~ Thes~ result-~ indicat~ that marrow progenitors maintained and retrovirally infected in L~MCs can engra~t in the face of a presumably ~trong endogenous recovery of marrow during the pancytopenic postirradiation phase.
2Q~8~'~
_ 30 - 8~276 T5bb a ~oa d P~ ~ h ~ eUb Al~ ~ T~l~ cO LT~g Cdb T _le~ M~ a~ _ w ~ w 1~10 N~ 1~10 ND 1 3 1.10 l/tll 1 Nl:l 0.1~1 V10 O.t~ 0 0.1-1 0 0.1~1 NO Ø1-1 9/30 Q1.1 Nl~
7 0.1~1 1/3D
0.014.1 31~a 0.1.1 12 0.1~ V~7 ~ 1 0.1-1 3/10 0.~1 14 0.1-1 0131~
1~1 Q~-t 019 7 0.1~1 Vl~ 0.1~1 ~1 Q1~1 3/11 7ll~ d ~ T ~ ~ h ma _ q ~ Al~_ d r~ 9#p~ h ON/~ o~l bam 1~ to al o o 3 1~ 0 0 12 0 1:1 1~ 0 0 ~I 12 0 0 PA31t~2 19' N~o N O
_ _ m~
2 ~
Figures 7a, 7b and 7c show that tha levels of engra~tment observed equalled or surpassed thoss obtained previously when dogs were sub~ecte.d to marrow ablative therapy prior to the infusion of miarked cells.
Th~ present results indicate that; (1) LTMC cells can reconstitute the hematopoietic systems of large animals; ~2) Marrow ablative conditioning is not necessary ~or engraftment of LTMC cells and may, in fact, cQmpromise engraft~ent by upregulating endogenous hematopoiesis; (3) Only a few stem cells are cycling at any given time in large mammals; an~ (4) I~L vitro activated st~m cells complete normal differentiation and proliferation programs when returned to the ~nL_YiYQ
microenvironments ~rom whence they came.
While the inv~ntion has been dèscribed in detail and with reference to specific embodiments thereo~, it will be apparent to one skilled in the art that various changes and modifications can be mada therein without deporting from the spirit and scope o~ the invention.
.
Claims (28)
1. A method of effecting transfer of a gene into gene transfer target cells comprising establishing a long term culture containing said cells in a suitable medium and exposing said culture to multiple, periodic infections of a gene transfer vehicle containing said gene to provide the gene-containing target cell.
2. A method as claimed in Claim 1 wherein said gene transfer target cell is a regenerative stem cell.
3. A method as claimed in Claim 2 wherein said regenerative stem cell is selected from the group consisting of hematopoietic stem cells, epithelial stem cells, exocrine gland stem cells, endocrine glad stem cells, hepatocyte stem cells, pancreatic stem cells, neuroendocrine stem cells, connective tissue stem cells, stromal stem cells fibroblast stem cells, mesenchymal stem cells, adipose cell stem cells, mammary gland stem cells, reticuloendothelial stem cells, lipid stem cells, chondrocyte stem cells, osteprogenitor stem cells, osteocyte stem cells, muscle fiber stem cells, neuronal stem cells, epidermal stem cells, kertinocyte stem cells, Langerhans stem cells, melanocyte stem cells, sebaceous gland stem cells, sweat gland stem cells, mucous stem cells, serous cell stem cells, odontoblast stem cells, islet of Langerhans stem cells, alveolar stem cells and retinal stem cells.
4. A method as claimed in Claim 3 wherein said stem cell is a hematopoietic stem cell.
5. A method as claimed in Claim 1 wherein said gene transfer target cells is selected from the hierarchy of committed cells that result in terminally differentiated mast cells, eosinophils, plasma cells, lymphoctyes, monoblasts, promonocytes, monocytes, macrophages, basophils neutrophils, erythrocytes, granulocytes, stromal cells, megakaryocytes, platelets, osteoblasts, osteocytes, osteoclasts, fibroblasts, epithelial cells, hepatocytes, pancreatic cells, neuroendocrine cells, connective tissue cells, mesenchymal cells, adiposytes, mammary gland cells, reticuloendothelial cells, lipid cells, chondrocytes, muscle cells, neurons, epidermal cells, Langerhans cells, melanocytes, gland cells, mucous cells, serous cells, odontoblasts, islet of Langerhans cells, alveolar cells and retinal cells.
6. A method as claimed in Claim 1 wherein said gene is an antibiotic resistant gene.
7. A method as claimed in Claim 1 further comprising multiple, periodic partial substitution of said medium.
8. A method as claimed in Claim 7 comprising:
establishing said culture in a first suitable medium;
exposing said culture to a first infection of said gene transfer vehicle;
incubating said infected culture for a sufficient period of time to effect cell proliferation and in vitro cell renewal;
substituting a portion of said first suitable medium with a second suitable medium to provide a first resultant medium;
exposing said culture to a second infection of said gene transfer vehicle;
further incubating said infected culture for a sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said gene-containing target cell.
establishing said culture in a first suitable medium;
exposing said culture to a first infection of said gene transfer vehicle;
incubating said infected culture for a sufficient period of time to effect cell proliferation and in vitro cell renewal;
substituting a portion of said first suitable medium with a second suitable medium to provide a first resultant medium;
exposing said culture to a second infection of said gene transfer vehicle;
further incubating said infected culture for a sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said gene-containing target cell.
9. A method as claimed in Claim 8 further comprising, prior to recovering said gene-containing target cell, substituting a portion of said first resultant medium with a third suitable medium to provide a second resultant medium; exposing said culture to a third infection of said gene transfer vehicle; and further incubating said culture for a sufficient period of time to effect yet further cell proliferation and in vitro cell renewal.
10. A method as claimed in Claim 8 wherein said second suitable medium is a non-gene transfer vehicle-containing medium.
11. A method as claimed in Claim 9 wherein said second suitable medium and/or said third suitable medium is a non-gene transfer vehicle-containing medium.
12. A method as claimed in Claim 8 or Claim 9 wherein said sufficient periods of time to effect cell proliferation are at least 3 days.
13. A method as claimed in Claim 8 or Claim 9 wherein said culture is exposed to said second infection and/or said third infection with said gene transfer vehicle at least 10 hours after the provision of said first and said second resultant medium.
14. A method as claimed in Claim 1 wherein said gene transfer vehicle is a vector selected from the group consisting of a retrovirus, virus, plasmid, artificial and chromosome and oligonucleotide.
15. A gene-containing target cell produced by a method as claimed in any one of Claims 1 - 14.
16. A pharmaceutical composition comprising a gene-containing target cells as claimed in claim 15 and a pharmaceutically acceptable carrier therefor.
17. A method of treating a genetically deficient living organism with a desired gene comprising administering to said organism an effective amount of said gene-containing target cell as claimed in Claim 15 or a pharmaceutical composition comprising said gene-containing target cell.
18. A method as claimed in Claim 17 wherein said living organism has a disease caused by a deficiency or resultant defect of said desired gene.
19. A method as claimed in Claim 18 wherein said desired gene in said gene-containing target cells is provided to enhance the genetic material of said organism.
20. A method as claimed in any one of Claims 18 or 19 wherein said living organism is selected from the group consisting of bacteria, fungi, plants and animals.
21. Use of a gene-containing target cell as claimed in Claim 15 in the manufacture of a medicament.
22. A method for activating quiescent repopulating stem cells in vitro to provide activated stem cells which maintain their activity in vivo comprising establishing a long term culture containing said quiescent stem cells in a suitable medium and multiple, periodic partial substitution of said medium to provide said activated stem cell and recovering said activated stem cell.
23. A method as claimed in claim 22 comprising establishing said culture in a first suitable medium; incubating said culture for a first sufficient period of time to effect cell proliferation and in vitro cell renewal;
substituting a portion of said first suitable medium with a second suitable medium to provide a first resultant medium; further incubating said culture for a second sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said activated stem cell.
substituting a portion of said first suitable medium with a second suitable medium to provide a first resultant medium; further incubating said culture for a second sufficient period of time to effect further cell proliferation and in vitro cell renewal; and recovering said activated stem cell.
24. A method as claimed in claim 23 further comprising, prior to recovering said activated stem cell, substituting a portion of said first resultant medium with a third suitable medium to provide a second resultant medium; and further incubating said culture for a third sufficient period of time to effect yet further cell proliferation and in vitro cell renewal.
25. A method as claimed in claim 23 or claim 24 wherein said first, said second and/or said third sufficient periods of time to effect cell proliferation is at least 3 days.
26. Activated stem cells as produced by the method as claimed in any one of claims 22 to 25.
27. Method of enhancing the proliferation of pluripotent stem cells in vivo in a mammal comprising administering activated stem cells as claimed in claim 15 or in claim 26 to said mammal and allowing proliferation of said cells in said mammal in the absence of ablative marrow conditioning.
28. Method of claim 27 wherein said ablative marrow conditioning is selected from chemotherapy and irradiation to effect killing of abnormal cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2086844 CA2086844A1 (en) | 1993-01-07 | 1993-01-07 | Method and product of gene transfer into cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2086844 CA2086844A1 (en) | 1993-01-07 | 1993-01-07 | Method and product of gene transfer into cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2086844A1 true CA2086844A1 (en) | 1994-07-08 |
Family
ID=4150950
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2086844 Abandoned CA2086844A1 (en) | 1993-01-07 | 1993-01-07 | Method and product of gene transfer into cells |
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| CA (1) | CA2086844A1 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996015813A1 (en) * | 1994-11-23 | 1996-05-30 | University Of Massachusetts Medical Center | Cell compositions for use in transplantation and ex vivo gene therapy |
| WO1997048815A2 (en) * | 1996-06-17 | 1997-12-24 | Novartis Ag | Method for enhancing gene transfer |
| WO2001021184A1 (en) * | 1999-09-23 | 2001-03-29 | An-Go-Gen Inc. | Cell-based gene therapy for the pulmonary system |
| US6482406B1 (en) | 1999-03-26 | 2002-11-19 | Duncan J. Stewart | Cell-based gene therapy for the pulmonary system |
| US6592864B1 (en) | 1998-03-27 | 2003-07-15 | Duncan John Stewart | Cell-based gene therapy |
| US9585916B2 (en) | 1999-03-26 | 2017-03-07 | Northern Therapeutics Inc. | Cell based therapy for the pulmonary system |
-
1993
- 1993-01-07 CA CA 2086844 patent/CA2086844A1/en not_active Abandoned
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5665350A (en) * | 1994-11-23 | 1997-09-09 | University Of Massachusetts Medical Center | Cell cycle dependent transplantation and ex vivo gene therapy |
| US6068836A (en) * | 1994-11-23 | 2000-05-30 | University Of Massachusetts | Cell compositions for use in transplantation |
| WO1996015813A1 (en) * | 1994-11-23 | 1996-05-30 | University Of Massachusetts Medical Center | Cell compositions for use in transplantation and ex vivo gene therapy |
| WO1997048815A2 (en) * | 1996-06-17 | 1997-12-24 | Novartis Ag | Method for enhancing gene transfer |
| WO1997048815A3 (en) * | 1996-06-17 | 1998-03-26 | Ciba Geigy Ag | Method for enhancing gene transfer |
| US5928638A (en) * | 1996-06-17 | 1999-07-27 | Systemix, Inc. | Methods for gene transfer |
| US10660922B2 (en) | 1998-03-27 | 2020-05-26 | Northern Therapeutics Inc. | Cell-based therapy for the pulmonary system |
| US11654162B2 (en) | 1998-03-27 | 2023-05-23 | Northern Therapeutics, Inc. | Cell-based therapy for the pulmonary system |
| US10828336B2 (en) | 1998-03-27 | 2020-11-10 | Northern Therapeutics Inc. | Cell-based therapy for the pulmonary system |
| US6592864B1 (en) | 1998-03-27 | 2003-07-15 | Duncan John Stewart | Cell-based gene therapy |
| US9585916B2 (en) | 1999-03-26 | 2017-03-07 | Northern Therapeutics Inc. | Cell based therapy for the pulmonary system |
| US9827270B2 (en) | 1999-03-26 | 2017-11-28 | Northern Therapeutics, Inc. | Cell-based therapy for the pulmonary system |
| US6482406B1 (en) | 1999-03-26 | 2002-11-19 | Duncan J. Stewart | Cell-based gene therapy for the pulmonary system |
| WO2001021184A1 (en) * | 1999-09-23 | 2001-03-29 | An-Go-Gen Inc. | Cell-based gene therapy for the pulmonary system |
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