HMG 1-Effected Gene Transfer Using Cell Cultures and Tissue Explants Xenografted in Immunodeficient Mice

ABSTRACT Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contained the cDNA of the human α1-antitrypsin (hAAT) gene linked to the Rous sarcoma virus promoter was generated and administered systemically to mice. The level and duration of hAAT gene expression was similar to that achieved with an hAd counterpart in both immunocompetent and immunodeficient mice. However, the tissue distribution of the OAV vector differed from that observed for hAd vectors in that the liver was not the dominant target. Significantly, we demonstrated efficient gene transfer with the OAV vector into mice immunized with hAd vectors and vice versa. We also confirm that the immune response to a transgene product can prevent its functional expression following sequential application of a vector. Our results suggest a possible solution to endemic humoral immunity against currently used hAd vectors and should therefore have an impact on the design of improved gene therapy protocols utilizing adenovirus vectors.

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Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

Blood ◽

10.1182/blood.v86.1.101.bloodjournal861101 ◽

1995 ◽

Vol 86 (1) ◽

pp. 101-110 ◽

Cited By ~ 134

Author(s):

JA Nolta ◽

EM Smogorzewska ◽

DB Kohn

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Peripheral Blood ◽

Retroviral Vectors ◽

Optimal Conditions ◽

Hematopoietic Progenitors ◽

Immunodeficient Mice ◽

Stromal Support ◽

Colony Forming Assay ◽

Mobilized Peripheral Blood

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long- lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.

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Direct in vivo adenovirus-mediated gene transfer to salivary glands

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.266.6.g1146 ◽

1994 ◽

Vol 266 (6) ◽

pp. G1146-G1155 ◽

Cited By ~ 6

Author(s):

A. Mastrangeli ◽

B. O'Connell ◽

W. Aladib ◽

P. C. Fox ◽

B. J. Baum ◽

...

Keyword(s):

Salivary Gland ◽

Gene Transfer ◽

Salivary Glands ◽

De Novo ◽

Recombinant Adenovirus ◽

Salivary Gland Cell ◽

Immunodeficient Mice ◽

Ductal Cells ◽

Adenovirus Vectors

Gene transfer to the salivary glands holds the potential for the therapy of salivary gland disorders and for delivery of therapeutic proteins to the mouth and upper gastrointestinal tract. Administration of the recombinant adenovirus vectors Ad.RSV beta gal [coding for the intracellular protein beta-galactosidase (beta-Gal)] and Ad alpha 1AT [coding for human alpha 1-antitrypsin (alpha 1-AT), a secreted protein] to salivary gland cell lines in vitro demonstrated exogenous gene expression. Retrograde ductal injection of the Ad.RSV beta gal vector to rat salivary glands in vivo resulted in beta-Gal expression in acinar and ductal cells. Exposure of submandibular glands in vivo to Ad alpha 1AT resulted in expression of alpha 1-AT mRNA transcripts, de novo synthesis of alpha 1-AT, and secretion in the saliva. To evaluate the feasibility of adenovirus-mediated gene transfer to human glands, human minor salivary glands were infected ex vivo with Ad.RSV beta gal, and implanted into severe combined immunodeficient mice. Evaluation of the human tissue demonstrated beta-Gal activity. These observations demonstrate that adenovirus vectors are capable of direct delivery of genes to the salivary glands, suggesting a variety of possible gene therapy applications.

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