Douglas R. Kennedy

Severe Papillomavirus Infection Progressing to Metastatic Squamous Cell Carcinoma in Bone Marrow-Transplanted X-Linked SCID Dogs

ABSTRACT Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain (γc) gene and is identical clinically and immunologically to human XSCID, making it a true homologue of the human disease. Bone marrow-transplanted (BMT) XSCID dogs not only engraft donor T cells and reconstitute normal T-cell function but, in contrast to the majority of transplanted human XSCID patients, also engraft donor B cells and reconstitute normal humoral immune function. Shortly after our initial report of successful BMT of XSCID dogs, it soon became evident that transplanted XSCID dogs developed late-onset severe chronic cutaneous infections containing a newly described canine papillomavirus. This is analogous to the late-onset cutaneous papillomavirus infection recently described for human XSCID patients following BMT. Of 24 transplanted XSCID dogs followed for at least 1 year post-BMT, 71% developed chronic canine papillomavirus infection. Six of the transplanted dogs that developed cutaneous papillomas were maintained for >3 1/2 years post-BMT for use as breeders. Four of these six dogs (67%) developed invasive squamous cell carcinoma (SCC), with three of the dogs (75%) eventually developing metastatic SCC, an extremely rare consequence of SCC in the dog. This finding raises the question of whether SCC will develop in transplanted human XSCID patients later in life. Canine XSCID therefore provides an ideal animal model with which to study the role of the γc-dependent signaling pathway in the response to papillomavirus infections and the progression of these viral infections to metastatic SCC.

Intravenous injection of a foamy virus vector to correct canine SCID-X1

Current approaches to hematopoietic stem cell (HSC) gene therapy involve the collection and ex vivo manipulation of HSCs, a process associated with loss of stem cell multipotency and engraftment potential. An alternative approach for correcting blood-related diseases is the direct intravenous administration of viral vectors, so-called in vivo gene therapy. In this study, we evaluated the safety and efficacy of in vivo gene therapy using a foamy virus vector for the correction of canine X-linked severe combined immunodeficiency (SCID-X1). In newborn SCID-X1 dogs, injection of a foamy virus vector expressing the human IL2RG gene resulted in an expansion of lymphocytes expressing the common γ chain and the development of CD3(+) T lymphocytes. CD3(+) cells expressed CD4 and CD8 coreceptors, underwent antigen receptor gene rearrangement, and demonstrated functional maturity in response to T-cell mitogens. Retroviral integration site analysis in 4 animals revealed a polyclonal pattern of integration in all dogs with evidence for dominant clones. These results demonstrate that a foamy virus vector can be administered with therapeutic benefit in the SCID-X1 dog, a clinically relevant preclinical model for in vivo gene therapy.

Ex Vivo γ-Retroviral Gene Therapy of Dogs with X-linked Severe Combined Immunodeficiency and the Development of a Thymic T Cell Lymphoma

We have previously shown that in vivo γ-retroviral gene therapy of dogs with X-linked severe combined immunodeficiency (XSCID) results in sustained T cell reconstitution and sustained marking in myeloid and B cells for up to 4 years with no evidence of any serious adverse effects. The purpose of this study was to determine whether ex vivo γ-retroviral gene therapy of XSCID dogs results in a similar outcome. Eight of 12 XSCID dogs treated with an average of dose of 5.8 × 10(6) transduced CD34(+) cells/kg successfully engrafted producing normal numbers of gene-corrected CD45RA(+) (naïve) T cells. However, this was followed by a steady decrease in CD45RA(+) T cells, T cell diversity, and thymic output as measured by T cell receptor excision circles (TRECs) resulting in a T cell lymphopenia. None of the dogs survived past 11 months post treatment. At necropsy, few gene-corrected thymocytes were observed correlating with the TREC levels and one of the dogs was diagnosed with a thymic T cell lymphoma that was attributed to the gene therapy. This study highlights the outcome differences between the ex vivo and in vivo approach to γ-retroviral gene therapy and is the first to document a serious adverse event following gene therapy in a canine model of a human genetic disease.

Keratinocyte Antiviral Response to Poly(dA:dT) Stimulation and Papillomavirus Infection in a Canine Model of X-Linked Severe Combined Immunodeficiency

X-linked severe combined immunodeficiency (XSCID) is caused by a genetic mutation within the common gamma chain (γc), an essential component of the cytokine receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT) to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV) infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT) and canine papillomavirus-2 (CPV-2) upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG) on poly(dA:dT) stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT) as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients.