There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1-7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

In this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7,690,000 (7.69 ؋ 10 6 ) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 ؋ 10 6 IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of >20 mg/dl and protein at concentrations of >9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log 10 standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log 10 IU/ml) 95% of the time in clinically stable individuals.Hepatitis C virus (HCV) infection is a major health care problem, with an estimated 4 million individuals infected in the United States (1) and 170 million infected worldwide (26). Approximately 70% of those infected will develop chronic HCV infection, which is a leading cause of chronic liver disease (13). Interferon monotherapy was the first antiviral regimen approved for treatment of chronic HCV infection. Sustained response rates were low (8 to 12%) (3), but newer antiviral treatments (interferon plus ribavirin, peginterferon plus ribavirin) have improved the response rates, particularly for patients infected with non-1 genotypes.Quantitative HCV RNA results have been used prior to initiating interferon-based anti-HCV treatment to assess the likelihood of sustained virological response, defined as a negative result in a qualitative HCV RNA assay 6 months after the end of treatment. HCV RNA levels or changes from baseline have also been used early in treatment to attempt to accurately predict response to treatment (6,10,14,15,18,20,27).The performance characteristics of an assay, i.e., its accuracy, reproducibility, and predictive values, determine the interpretation and utility of the assay's results (12, 18). The VERSANT HCV bDNA Assay, utilizing a signal amplification technique, is a fundamentally simple hybridization-based procedure involving unique nucleic acid probes directed to highly co...

Extrapolation of an understanding regarding hematopoiesis and, in particular, hematopoietic stem cells (HSCs) from rodent models or in vitro human cell models to applications in humans has proven very difficult. This is not surprising, given the differences between rodent and human hematopoietic physiology and the lack of true in vitro assays for HSCs. Therefore, translational preclinical development of genetic and cellular therapies is dependent on the utilization of practical and well-defined large animal models. This chapter will introduce the most commonly used model species, including macaques, baboons, dogs, cats, and sheep, and explain the particular advantages and limitations of each. Specific protocols for the support of macaques through ablative cell and gene therapy procedures will be included to introduce investigators to the types of resources and support required to maintain a large animal facility dedicated to high-intensity experimentation, and also to introduce investigators to the types of procedures that are possible.

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An understanding of the number and contribution of individual pluripotent hemato-poietic stem cells (HSCs) to the formation of blood lineages has important clinical implications for gene therapy and stem cell transplantation. We have been able to efficiently mark rhesus macaque long-term repopulating stem and progenitor cells with retroviral vectors, and track their in vivo contributions to hematopoi-esis using the linear amplification mediated -polymerase chain reaction (LAM-PCR) technique of insertion site analysis. We assessed the impact of busulfan on contributions of individual retrovirally marked clones to hematopoiesis. There were 2 macaques that received transplants of retrovirally transduced CD34 cells 2 years previously that were then treated with 4 mg/kg busulfan. Despite only transient and mild suppression of peripheral blood counts, the numbers of individual stem/progenitor clones contributing to granulocyte production decreased dramatically , by 80% in the first monkey and by 60% in the second monkey. A similar impact was seen on clones contributing to T cells. The clone numbers recovered gradually back toward baseline by 5 months following busulfan in the first monkey and by 3 months in the second monkey, and have remained stable for more than one year in both animals. Tracking of individual clones with insertion-site-specific primers suggested that clones contributing to hematopoiesis prior to busulfan accounted for the majority of this recovery, but that some previously undetected clones began to contribute during this recovery phase. These results indicate that even low-dose busul-fan significantly affects stem and progenitor cell dynamics. The clonal diversity of hematopoiesis was significantly decreased after even a single, clinically well-tolerated dose of busulfan, with slow but almost complete recovery over the next several months, suggesting that true long-term repopulating stem cells were not permanently deleted. However, the prolonged period of suppression of many clones suggests that transplanted HSCs may have a marked competitive advantage if they can engraft and proliferate during this time period, and supports the use of this agent in nonmyeloablative regimens (Blood.