Margaret Brzezinski

Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression

Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

Long-Term Erythropoietin Gene Expression from Transduced Cells in Bioisolator Devices

Recombinant erythropoietin (EPO) is widely administered for long-term treatment of anemia associated with renal failure and other chronic diseases. The ability to deliver EPO by gene therapy would have clinical and economic benefit. We compared autologous and allogeneic transduced primary vascular smooth muscle cells for their ability to provide sustained EPO gene expression when encapsulated in TheraCyte devices implanted subcutaneously (SQ) or intraperitoneally (IP) in rats. Cells were transduced with retrovirus vector LrEpSN encoding rat EPO cDNA. Rats that received either autologous or allogeneic transduced cells showed elevated hematocrits (HCTs) ranging from 50 to 79% that were sustained for more than 12 months. The HCT of control rats remained at baseline (45.8%). Rats that received second SQ implants of either autologous or allogeneic cells showed elevations in hematocrit that were sustained for up to 12 months, suggesting the absence of immunological responses to transduced cells or implant material. All experimental groups had statistically significant elevated HCT (p < 0.001) when compared with controls. Both SQ and IP implantation were equally effective in delivering EPO long term. There were no significant differences in white blood cell (WBC) or platelet (PLT) values between treated and control animals. Implantation of TheraCyte devices was well tolerated and histological evaluation of the devices up to 12 months after surgery revealed a high degree of vascularization and no evidence of host immune response. TheraCyte devices offer a simple and safe gene delivery system that provides sustained therapeutic gene expression, permit removal and implantation of new devices, and do not require immunosuppression of the host.

Prolonged survival and improved glycemia in BioBreeding diabetic rats after early sustained exposure to glucagon-like peptide 1

Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune disease that results in complete destruction of islets and insulin dependency for life. Glucagon‐like peptide 1 (GLP‐1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. We hypothesized that the expression of GLP‐1 before disease onset would increase islet mass, delay diabetes and prolong survival of BB rats.

G-CSF-lentivirus administration in rats provided sustained elevated neutrophil counts and subsequent EPO-lentivirus administration increased hematocrits

Towards gene therapy treatment of patients with neutropenia, characterized by neutrophil counts < 500 cells/µl, we investigated the ability of lentivirus vectors to provide sustained granulocyte colony‐stimulating factor (G‐CSF) delivery and to permit transgene expression from a second virus administration in a preclinical rat model.

Sustained elevation of neutrophils in rats induced by lentivirus-mediated G-CSF delivery.

Patients with severe chronic and cyclic neutropenia, characterized by neutrophil numbers <500 cells/µl, are treated daily with recombinant granulocyte colony‐stimulating factor (G‐CSF). As an alternative delivery approach we investigated the ability of lentivirus vectors to provide sustained G‐CSF expression.

459. Long-Term Treatment of Canine Cyclic Neutropenia by G-CSF-Lentivirus

Cyclic neutropenia (CN) occurs both in man and grey collie dogs and is characterized by recurrent periods of neutropenia leading to bacterial infections and shortened life expectancy. Daily rG-CSF provides therapy for patients and dogs. To treat CN by gene therapy we constructed a VSV-G pseudotyped lentivirus encoding canine G-CSF cDNA regulated by CMV promoter, cPPT from HIV 1 and PRE from human hepatitis B virus. At 7 weeks of age when an affected grey collie dog was weaned, serial blood counts were monitored. These data showed the dogs absolute neutrophil counts (ANC) were cycling and observations over 4 cycles established a 12 day periodicity. The ANC mean|[plusmn]|SD during nadirs and peaks were 560|[plusmn]|787 cells/|[mu]|l and 10,760|[plusmn]|1,120 cells/|[mu]|l respectively and overall counts were 5,240|[plusmn]|4,790 cells/|[mu]|l. Having established that neutrophils were cycling, recombinant canine G-CSF was administered subcutaneously at a dose of 1.5 |[mu]|g/kg per weekday for two months. Although ANC continued to cycle, we observed significant increases in neutrophils both at the nadirs and the peaks of cycles (p<0.0001) with mean values of 4,860|[plusmn]|2,670 cells/|[mu]|l and 28,870|[plusmn]|9,830 cells/|[mu]|l respectively. Over this period the mean count was 17,820|[plusmn]|11,100 cells/|[mu]|l. After demonstrating that rG-CSF elevated neutrophil production cytokine administration was stopped and we administered IM 2|[times]|109 infectious units(IU) of G-CSF-lentivirus. The dog showed no ill effects to the virus injections. Serial monitoring of blood cells showed elevated ANC for over 17 months with mean value of 29,230|[plusmn]|12,930 cells/|[mu]|l (n=217) that was significantly increased over both no treatment and rG-CSF treatment (p<0.0001). The increases in ANC from recombinant G-CSF and lentivirus administration were associated with absence of clinical signs of infection and fever.