Tanya H. Burkholder

Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

Health Evaluation of Experimental Laboratory Mice

Good science and good animal care go hand in hand. A sick or distressed animal does not produce the reliable results that a healthy and unstressed animal produces. This unit describes the essentials of assessing mouse health, colony health surveillance, common conditions, and determination of appropriate endpoints. Understanding the health and well‐being of the mice used in research enables the investigator to optimize research results and animal care. Curr. Protoc. Mouse Biol. 2:145‐165

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Gene Therapy of Canine Leukocyte Adhesion Deficiency Using Lentiviral Vectors With Human CD11b and CD18 Promoters Driving Canine CD18 Expression

To identify cellular promoters in a self-inactivating (SIN) lentiviral vector that might be beneficial in treating children with leukocyte adhesion deficiency type 1 (LAD-1), we tested lentiviral vectors with human CD11 and CD18 leukocyte integrin proximal promoter elements directing expression of canine CD18 in animals with canine LAD (CLAD). Lentiviral vectors with either the human CD11b (637 bp) proximal promoter or the human CD18 (1,060 bp) proximal promoter resulted in the highest percentages of CD18(+) CLAD CD34(+) cells in vitro. Subsequently, two CLAD dogs were infused with autologous CD34(+) cells transduced with the hCD11b (637 bp)-cCD18 vector, and two CLAD dogs were infused with autologous CD34(+) cells transduced with the hCD18 (1,060 bp)-cCD18 vector. Each dog received a nonmyeloablative dose of 200 cGy total body irradiation (TBI) before the infusion of transduced cells. The two CLAD dogs treated with the hCD18 (1,060 bp)-cCD18 vector, and one of the two dogs treated with the hCD11b (637 bp)-cCD18 vector, had reversal of the CLAD phenotype. These studies using endogenous leukocyte integrin proximal promoters represent an important step in the development of gene therapy for children with LAD-1.

Treatment of canine leukocyte adhesion deficiency by foamy virus vectors expressing CD18 from a PGK promoter

Proto-oncogene activation caused by retroviral vector integration can cause malignancies in gene therapy trials. This has led investigators to search for less genotoxic vectors with minimal enhancer activity and a decreased risk of influencing neighboring chromosomal gene expression after integration. We previously showed that foamy virus (FV) vectors expressing the canine CD18 gene from an internal murine stem cell virus (MSCV) promoter could cure canine leukocyte adhesion deficiency (LAD). Here, we have repeated these studies using a FV vector expressing canine CD18 from a phosphoglycerate kinase (PGK) gene promoter. In vitro analysis showed that this vector did not contain an enhancer that activated neighboring genes, and it expressed CD18 efficiently in canine neutrophils and CD34+ cells. However, dogs that received hematopoietic stem cells transduced with the PGK-CD18 vector continued to suffer from LAD, and sometimes died prematurely of the disease. These studies show that the PGK promoter cannot effectively replace the MSCV promoter in CD18-expressing FV vectors, and they suggest that vectors containing a strong promoter–enhancer may be necessary for the treatment of human LAD.

Lentiviral vectors incorporating a human elongation factor 1α promoter for the treatment of canine leukocyte adhesion deficiency

Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1α (EF1α) promoter (EF1αL, 1189 bp and EF1αS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1αS vector resulted in the highest levels of canine CD18 expression in CLAD CD34+ cells in vitro. Subsequently, autologous CD34+ bone marrow cells from four CLAD pups were transduced with the EF1αS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18+ neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1αS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18+ neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.

Gene Therapy for Canine Leukocyte Adhesion Deficiency with Lentiviral Vectors Using the Murine Stem Cell Virus and Human Phosphoglycerate Kinase Promoters

Children with leukocyte adhesion deficiency type 1 (LAD-1) and dogs with canine LAD (CLAD) develop life-threatening bacterial infections due to mutations in the leukocyte integrin CD18. Here, we compared the human phosphoglycerate kinase (hPGK) promoter to the murine stem cell virus (MSCV) promoter/enhancer in a self-inactivating HIV-1-derived lentiviral vector to treat animals with CLAD. Four CLAD dogs were infused with CD34(+) cells transduced with the hPGK vector, and two CLAD dogs received MSCV vector-transduced CD34(+) cells. Infusions were preceded by a nonmyeloablative dose of 200 cGy total body irradiation. Comparable numbers of transduced cells were infused in each group of animals. Only one of four CLAD animals treated with the hPGK-cCD18 vector had reversal of CLAD, whereas both MSCV-cCD18 vector-treated dogs had reversal of the phenotype. Correction of CLAD depends both upon the percentage of CD18(+) myeloid cells and the level of expression of CD18 on individual myeloid cells. In this regard, the hPGK promoter directed low levels of expression of CD18 on neutrophils compared to the MSCV promoter, likely contributing to the suboptimal clinical outcome with the hPGK vector.

The Rabbit as an Experimental Model

Publisher Summary This chapter demonstrates the continued high use of rabbits in the areas of atherosclerosis, ophthalmic disease, infectious disease, orthopedic research, and experimental neoplastic research. The chapter provides an overview of these conditions. The total number of rabbits used for research and testing in the United States has declined from a peak value of approximately 550,000 per year in the late 1980s to the current level of approximately 230,000. Several factors contribute to this decline and the most important being the rapid expansion of genetically engineered rodent models, which largely replace rabbits for investigation of many diseases such as candidiasis and variola. Even for conditions such as herpes simplex keratitis in which rabbits are still a classic and frequently used model, murine models have been developed to replace rabbits. These tests were developed in the 1960s and adopted by the Environmental Protection Agency to meet the testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act and the Toxic Substances Control Act. The albino rabbit remains the preferred species for both of these tests.

Biology and Diseases of Cats

Domestic cats (Felis cattus) comprise a small (2%) percentage of the non-rodent animals used in biomedical research. In 2011, 21,700 cats of a total 1,134,693 non-rodent animals were used in research (APHIS, 2011). According to the National Research Council Committee on Scientific and Humane Issues in the Use of Random Source Dogs and Cats in Research (National Research Council, 2009), peak use of cats occurred in 1974. Since that time, the number of cats used in research has fallen by 71%, with more than 98% of those cats being purpose bred for research. Cats are a U.S. Department of Agriculture (USDA) covered species with special housing requirements defined in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NRC, 2011). At the request of congress, a committee of experts formed by the National Research Council examined the use of random source dogs and cats and concluded that obtaining dogs and cats from Class B dealers is not necessary for NIH funded research (National Research Council, 2009). While the number of cats used in biomedical research has declined, cats continue to contribute uniquely to biomedical science and are valuable research model for several disciplines, including aspects of neurology involved in locomotion and spinal trauma, retrovirus and zoonotic disease research, and for developing therapeutic strategies for inherited diseases.

458. Reversal of Canine Leukocyte Adhesion Deficiency by Retroviral-Vector Mediated Gene Therapy with Non-Myeloablative Conditioning

Children with leukocyte adhesion deficiency or LAD suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD is due to heterogenous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency or CLAD, like children with LAD, also experience severe bacterial infections and typically die within the first few months of life. CLAD represents a disease-specific, large animal model for testing new therapeutic approaches for the human disease LAD. We tested a retroviral-vector mediated gene therapy approach in CLAD using a non-myeloablative conditioning regimen. Seven CLAD dogs received autologous, CD34+ gene-corrected cells following 200 cGy total body irradiation (TBI). CLAD CD34+ cells were pre-stimulated overnight with growth factors cIL-6, cSCF, hFlt3-L, and hTPO, then incubated with retroviral vector PG13/MSCV-cCD18 over 48 hours on recombinant fibronectin. Transduction of the CLAD CD34+ cells ranged from 15 to 33%. Following transduction, cells were re-infused (0.3 to 1.8 |[times]| 106 CD18+ cells per kg) after 200 cGy TBI. One group of 4 dogs received post-transplant immunosuppression consisting of cyclosporine and mycophenolate mofetil. A second cohort group of 3 dogs received no post-transplant immunosuppression. Peripheral blood samples were analyzed by flow cytometry for CD18 expression. At a mean time of 6 months post-gene transfer, the CD18+ gene-corrected leukocyte frequency in the peripheral blood ranged from 0.044% to a high of 3.15%. Five of the 7 CLAD dogs (3 with immunosuppression, 2 without immunosuppression) receiving CD18+ gene corrected cells have had significant improvement of their CLAD disease and are alive and well at 9 to13 months of age. These results contrast markedly with those seen in untreated CLAD dogs that die or are euthanized within the first few months of life due to intractable infection. Our studies indicate that a non-myeloablative regimen of 200 cGy TBI regimen facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the disease phenotype in CLAD, and that post-transplant immunosuppression is not required for the persistence of CD18 gene-corrected cells. These results provide support for the use of a non-myeloablative conditioning regimen prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.