Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning.

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity.

Long-Term Persistence of Anti-HIV Broadly Neutralizing Antibody-Secreting Hematopoietic Cells in Humanized Mice.

Broadly neutralizing antibodies (bNAbs) are among the most promising strategies to achieve long-term control of HIV-1 in the absence of combination antiretroviral therapy. Passive administration of such antibodies in patients efficiently decreases HIV-1 viremia, but is limited by the serum half-life of the protein. Here, we investigated whether antibody-secreting hematopoietic cells could overcome this problem.

Minimal conditioning in Fanconi anemia promotes multi-lineage marrow engraftment at 10-fold lower cell doses.

Background: Gene therapy approaches for the treatment of Fanconi anemia (FA) hold promise for patients without a suitably matched donor for an allogeneic bone marrow transplant. However, significant limitations include the collection of sufficient stem cell numbers from patients, the fragility of these cells during ex vivo manipulation, and clinically meaningful engraftment following transplantation. With these challenges in mind, we were interested in determining (i) whether gene-corrected cells at progressively lower numbers can successfully engraft in FA; (ii) whether low-dose conditioning facilitates this engraftment; and (iii) whether these cells can be selected for post-transplant.

HIV infection results in clonal expansions containing integrations within pathogenesis-related biological pathways.

The genomic integration of HIV into cells results in long-term persistence of virally infected cell populations. This integration event acts as a heritable mark that can be tracked to monitor infected cells that persist over time. Previous reports have documented clonal expansion in people and have linked them to proto-oncogenes; however, their significance or contribution to the latent reservoir has remained unclear.

In Vivo Murine-Matured Human CD3 Cells as a Preclinical Model for T Cell-Based Immunotherapies.

Adoptive cellular immunotherapy is a promising and powerful method for the treatment of a broad range of malignant and infectious diseases. Although the concept of cellular immunotherapy was originally proposed in the 1990s, it has not seen successful clinical application until recent years. Despite significant progress in creating engineered receptors against both malignant and viral epitopes, no efficient preclinical animal models exist for rapidly testing and directly comparing these engineered receptors.

In vivo protection of activated Tyr22-dihydrofolate reductase gene-modified canine T lymphocytes from methotrexate.

Background: Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment.

Stem cell selection in vivo using foamy vectors cures canine pyruvate kinase deficiency.

Background: Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies.

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates.

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle.

A retroviral vector common integration site between leupaxin and zinc finger protein 91 (ZFP91) observed in baboon hematopoietic repopulating cells.

Objective: Retroviral vector proviruses can lead to aberrant expression of nearby genes in hematopoietic repopulating cells, leading to an over-representation of clones with dysregulated genes that affect hematopoiesis. Common integration sites (CISs) identified using the vector provirus as a molecular tag can be used to identify these genes. Here we characterized a retroviral CIS observed at high frequency in baboon hematopoietic repopulating cells that has not been described previously.

Cocal-pseudotyped lentiviral vectors resist inactivation by human serum and efficiently transduce primate hematopoietic repopulating cells.

Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR).

Stable marking and transgene expression without progression to monoclonality in canine long-term hematopoietic repopulating cells transduced with lentiviral vectors.

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients.

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS).

Foamy and lentiviral vectors transduce canine long-term repopulating cells at similar efficiency.

Foamy viral vectors and lentiviral vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy because they both efficiently transduce stem cells using rapid ex vivo transduction protocols designed to maintain engraftment potential. Here we directly compared the ability of foamy and lentiviral vectors to transduce long-term hematopoietic repopulating cells in the dog model, using a competitive repopulation assay with vectors that express enhanced yellow or green fluorescent proteins (EY/GFP). Mobilized canine peripheral blood CD34(+) cells were divided into two fractions and exposed to either foamy (EGFP) or lentiviral (EYFP) vectors at a multiplicity of infection of 5 in an 18-hr transduction protocol and then reinfused after conditioning with 920 cGy of total body irradiation.