Robert E. Richard

American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on Retroviral-Mediated Gene Transfer to Hematopoietic Stem Cells

Gene transfer using retroviral vectors has been under clinical study for more than 12 years1. Many studies have targeted hematopoietic stem cells (HSCs) as a potentially enduring and renewable source of gene-modified blood cells for the treatment of specific genetic diseases, cancer, leukemia, and HIV-1 infection2. Although initial studies were hampered by very low levels of gene transfer to HSCs, incremental progress has been realized in the efficiency of gene transfer to HSCs. These advances have culminated in the report of clinically significant restoration of immunity in patients with the X-linked form of severe combined immune deficiency (XSCID) by Alain Fischer, Marina Cavazzana-Calvo, and colleagues at the Hopital Necker Enfants Malades in Paris3. Their study and those conducted by Adrian Thrasher and colleagues at the Great Ormond Street Childrens Hospital in London for XSCID and by Claudio Bordignon and colleagues at the Hospital San Raffaele in Milan for children with SCID due to deficiency of adenosine deaminase (ADA) provide incontrovertible proof that gene therapy can ameliorate genetic diseases4,5.

In vivo selection using a cell-growth switch

A major obstacle to stem-cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant fraction of stem cells. One way to circumvent this obstacle is to use selection. Vectors containing two linked genes serve as the basis for selection, with one gene encoding a selectable product and the other, a therapeutic protein. Applying selection in vivo has the potential to bring a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a ‘cell-growth switch’, allowing a minor population of genetically modified cells to be inducibly amplified, thereby averting the risks associated with cytotoxic drugs. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy.

FGFR4 and its novel splice form in myogenic cells: interplay of glycosylation and tyrosine phosphorylation

The family of fibroblast growth factor receptors (FGFRs) is encoded by four distinct genes. FGFR1 and FGFR4 are both expressed during myogenesis, but whereas the function of FGFR1 in myoblast proliferation has been documented, the role of FGFR4 remains unknown. Here, we report on a new splice form of FGFR4 cloned from primary cultures of mouse satellite cells. This form, named FGFR4(‐16), lacks the entire exon 16, resulting in a deletion within the FGFR kinase domain. Expression of FGFR4(‐16) coincided with that of wild‐type FGFR4 in all FGFR4‐expressing tissues examined. Moreover, expression of both FGFR4 forms correlated with the onset of myogenic differentiation, as determined in mouse C2C12 cells and in the inducible myogenic system of 10T½‐MyoD‐ER cell line. Both endogenous and overexpressed forms of FGFR4 exhibited N‐glycosylation. In contrast to FGFR1, induced homodimerization of FGFR4 proteins did not result in receptor tyrosine phosphorylation. Surprisingly, coexpression of FGFR4 forms and a chimeric FGFR1 protein resulted in FGFR4 tyrosine phosphorylation, raising the possibility that FGFR4 phosphorylation might be enabled by a heterologous tyrosine kinase activity. Collectively, the present study reveals novel characteristics of mouse FGFR4 gene products and delineates their expression pattern during myogenesis. Our findings suggest that FGFR4 functions in a distinctly different manner than the prototype FGFR during myogenic differentiation. J. Cell. Physiol. 215: 803–817, 2008.

Small-molecule-directed Mpl signaling can complement growth factors to selectively expand genetically modified cord blood cells

Efforts toward achieving gene therapy for blood disorders are plagued by low rates of gene transfer into hemopoietic stem cells. Recent studies suggest that this obstacle can be circumvented using selection. One way to achieve selection employs genes that encode receptor‐bearing fusion proteins capable of inducing cell growth in response to drugs called chemical inducers of dimerization (CIDs). We have previously shown that genetically modified marrow cells from mice can proliferate for up to a year in culture in response to CID‐initiated signals arising from the thrombopoietin receptor (mpl). The sustained growth observed in mouse hemopoietic cells results from an mpl‐induced self‐renewal of multipotential hemopoietic progenitor cells. In contrast, human hemopoietic cells proliferate only transiently in response to the mpl signal (from differentiation of transduced erythroid and megakaryocytic progenitors), while human myeloid progenitors fail to respond. Here, we show that myeloid progenitors from human cord blood can be induced to proliferate and/or differentiate in response to the mpl signal by providing additional signals via a combination of growth factors. These findings are relevant for the eventual clinical application of CID‐regulated cell therapy.

Lymphomatoid granulomatosis associated with azathioprine use for immune-mediated neuropathy

Lymphomatoid granulomatosis (LG) is a rare Epstein-Barr virus-driven lymphoproliferative disorder that generally arises in immunosuppressed patients and which can be life-threatening. Here we describe the development of pulmonary LG in a patient on long-term azathioprine for immune-mediated neuropathy. Although azathioprine carries a boxed warning for malignancy, its association specifically with LG, an otherwise rare entity, is poorly recognised. Early recognition of drug-induced LG is critical, since discontinuation of the offending agent, and implementation of effective therapy can provide rapid clinical benefit in some patients. In this case, rituximab was used as an effective treatment for LG, which also provided an additional benefit of controlling the patients underlying neuropathy. Further research is needed to identify vulnerable patients who are at high risk of developing drug-induced LG.

Multiple Myeloma and the Kidney

RG, a 60-year-old African-American man, presents to your clinic with a 6-week history of fatigue and right hip pain, which he attributes to a fall a few months ago. He has not seen a physician in years because he has felt well. However, he did visit the ER a week ago for his hip pain and labs showed a creatinine of 1.9 mg/dL. He has been taking 800–1,200 mg of ibuprofen daily for the last 2 weeks. His physical examination is notable for pain over the right hip. Laboratory studies show a creatinine of 4.6 mg/dL, calcium of 11 mg/dL, and hemoglobin of 7.2 g/dL. Plain films of the right hip show osteolytic lesions. Urine dipstick is negative for protein.

987. Foamy Virus Vectors That Block HIV Infection

Foamy virus (FV) vectors have been demonstrated to transduce human hematopoietic stem cells with a high frequency. We have developed FV vectors that can block HIV infection with the intention of developing a therapeutic vector. Additional features of FV vectors that support their use in clinical gene therapy include: a) no evidence of disease from the prototypic FV in infected humans and in non-human primates, b) reduced recombination potential with HIV in infected patients, c) FV vectors are self-inactivating vectors, and d) relative to lentivirus vectors, FV vector production does not share molecular pathways with HIV.