Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells.

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

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Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.838-846.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 838-846

Author(s):

R S McIvor ◽

M J Johnson ◽

A D Miller ◽

S Pitts ◽

S R Williams ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Adenosine Deaminase ◽

Base Pair ◽

Purine Nucleoside Phosphorylase ◽

Regulatory Elements ◽

Purine Nucleoside ◽

Northern Analysis ◽

Hematopoietic Stem ◽

Nucleoside Phosphorylase

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

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Genetic Manipulation of Hematopoietic Stem Cells in Adenosine Deaminase Deficiency

Purine and Pyrimidine Metabolism in Man VIII - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4615-2584-4_86 ◽

1995 ◽

pp. 395-400

Author(s):

David A. Williams ◽

Thomas Moritz

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Adenosine Deaminase ◽

Genetic Manipulation ◽

Adenosine Deaminase Deficiency ◽

Hematopoietic Stem

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Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Journal of Experimental Medicine ◽

10.1084/jem.174.4.837 ◽

1991 ◽

Vol 174 (4) ◽

pp. 837-845 ◽

Cited By ~ 33

Author(s):

F A Fletcher ◽

K A Moore ◽

M Ashkenazi ◽

P De Vries ◽

P A Overbeek ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Adenosine Deaminase ◽

Leukemia Inhibitory Factor ◽

Ex Vivo ◽

Retroviral Vector ◽

Inhibitory Factor ◽

Hematopoietic Stem ◽

Infection Efficiency

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

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