In patients with alpha 1-antitrypsin deficiency, the development of emphysema is believed to be caused by the unchecked action of proteases on lung tissue. We evaluated the feasibility, safety, and biochemical efficacy of intermittent infusions of alpha 1-antitrypsin in the treatment of patients with alpha 1-antitrypsin deficiency. Twenty-one patients were given 60 mg of active plasma-derived alpha 1-antitrypsin per kilogram of body weight, once a week for up to six months. After a steady state had been reached, the group had trough serum levels of alpha 1-antitrypsin of 126 +/- 1 mg per deciliter as compared with 30 +/- 1 mg per deciliter before treatment, and serum anti-neutrophil elastase capacities of 13.3 +/- 0.1 microM as compared with 5.4 +/- 0.1 microM. The alpha 1-antitrypsin level in the epithelial-lining fluid of the lungs was 0.46 +/- 0.16 microM before treatment, and the anti-neutrophil elastase capacity was 0.81 +/- 0.13 microM. Six days after infusion, alpha 1-antitrypsin levels (1.89 +/- 0.17 microM) and anti-neutrophil elastase capacities (1.65 +/- 0.13 microM) in the lining fluid were significantly increased (P less than 0.0001). Because of the chronicity of the disorder and the lack of sensitive measures of lung destruction, the clinical efficacy of this therapy could not be studied rigorously. No changes in lung function were observed in our patients over six months of treatment. The only important adverse reactions to the 507 infusions were four episodes of self-limited fever. This study demonstrates that infusions of alpha 1-antitrypsin derived from plasma are safe and can reverse the biochemical abnormalities in serum and lung fluid that characterize this disorder. Together with lifetime avoidance of cigarette smoking, replacement therapy with alpha 1-antitrypsin may be a logical approach to long-term medical treatment.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34+ cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

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Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell (HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells (STR), or the fibronectin fragment CH-296 (FN), and various cytokines such as flt3 ligand (FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and clinically feasible peripheral blood (PB) progenitor cell transduction methods. First, we compared transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor (SCF), and FLT (our best cytokine combination in prior studies) with a combination of megakaryocyte growth and development factor (MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10-15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.