The concept of cell-based therapy for AAT deficiency was initially demonstrated by Garver and coworkers, who employed a retrovirus to introduce human AAT cDNA into murine fibroblasts in culture prior to intraperitoneal transplantion into nude mice (7
). Recent advances in stem cell biology and the development of lentiviral vectors allow us to extend this approach to transplantable stem cells with well-characterized self-renewal and differentiation capacities.
Our results support a novel approach to the systemic delivery of AAT. This approach harnesses the considerable capacity of HSCs to expand in vivo and uses lentiviruses to integrate genes into the HSC genome. In doing so, we adapt our previously reported method for stem cell transduction to accomplish prolonged expression of circulating human AAT in vivo. This work suggests stem cells in general and HSCs in particular as potential targets for gene therapy of AAT deficiency.
HSCs transduced to overexpress AAT appear to retain their capacity for engraftment and long-term multilineage blood reconstitution. The ability of transduced HSCs to contribute to all peripheral blood cell lineages is particularly significant given recent publications regarding the putative role of AAT in the regulation of bone marrow cell populations (34
). AAT is produced by a variety of hematopoietic cell types in the bone marrow, and down-regulation of AAT expression in the marrow to allow protease-based cleavage of matrix anchors may be important for the release of hematopoietic progenitors into the peripheral blood (34
). If true, overexpression of AAT in the marrow could interfere with progenitor cell mobilization. We found that HSCs transduced to overexpress AAT contributed to all peripheral blood lineages, and the proportion of cells that were GFP+ remained constant as cells were mobilized from bone marrow to blood compartments. Although transduced (GFP+) cells competed well with untransduced (GFP-) cells in their ability to contribute to the peripheral blood over time, cytokine mobilization experiments would be required to properly test for any effect of AAT overexpression on the kinetics of blood progenitor mobilization from marrow.
Despite accomplishing transduction of the majority of the hematopoietic system, our method did not result in circulating hAAT levels in mice above the theoretical “protective threshold” of 800 μg/ml in human serum, the level thought to protect patients from developing emphysema (18
). This contrasts with previous studies in which transduction of muscle fibers with adeno-associated viral vectors (AAV) was able to achieve “protective” levels of circulating hAAT in mice (8
). The levels achieved may reflect intrinsic secretory limitations of hematopietic cell types compared with hepatocytes and myocytes, or may be a consequence of limited promoter activity levels in our studies. Low circulating levels do not appear to have resulted from either a cell-mediated or from a humoral immune response directed against the hAAT protein, in contrast to prior gene therapy approaches (8
). This result is consistent with the observation that HSC transplantation and resultant immune reconstitution typically abrogates humoral and cellular immunity against heterologous proteins expressed by the donor-derived stem cells.
Future studies employing lentiviral transduction of other progenitor cell populations, such as hepatic oval cells, muscle satellite cells, or embryonic stem cell–derived hepatocytes may reveal better targets for cell-based therapies of AAT deficiency and result in higher levels of AAT expression. Alternatively, evaluation of additional nonviral promoters may reveal candidates able to drive higher levels of gene expression. Indeed, in our studies, the highest level of hAAT expression in the plasma resulted from HSCs transduced with AAT under control of the human UBC promoter.
It is notable that estimated hAAT expression in the ELF exceeded plasma expression in some animals. This result raises the possibility that a local pool of transduced cells, such as alveolar macrophages, contributed to hAAT production in these recipients. A lung-localized hAAT-producing cell could be more effective than a distant one in suppressing protease activity in the lung. It is known that only a fraction of circulating AAT reaches the lung interstitium, where it purportedly exerts its protective benefit (18
). While the serum AAT threshold necessary to provide protection from lung damage is widely accepted to be approximately 800 μg/ml, the level required in the lung itself to provide this benefit is likely to be much lower (18
A significant potential limitation to our approach is the use of bone marrow transplant (BMT), a procedure that carries with it the risk of infection as well as complications resulting from chemo- or radiotherapy. However, nonmyeloablative BMTs using Busulfan or direct modulation of endogenous HSCs may offer a less-toxic alternative for patients undergoing HSC transplantation (22
). The use of integrating vectors, such as lentiviruses, to transduce stem cell populations raises the additional concern of the potential for oncogenesis. The likelihood of lentiviral-induced insertional mutagenesis may be reduced by decreasing the number of target cells subjected to proviral integration and the number of integrating events per cell. Previously published work (20
) has determined that HSC transduction with our protocol results in one to five proviral insertions per transduced cell. In contrast to protocols employing simple retroviral vectors in which millions of cells are typically transplanted (17
), our approach targets low numbers of cells (3,000), minimizing the statistical chance of insertional mutagenic events. Although experimental mice in this study and others had normal life spans and exhibited no evidence of leukemia (20
), the potential for lentiviruses and other complex retroviruses to cause insertional mutagenesis is an area that warrants further study. Finally, this method and others aimed solely at increasing circulating wild-type hAAT levels do not address the hepatotoxic effects of misfolded mutant AAT proteins. Therapies that incorporate silencing of mutant AAT genes are lacking and will need to be developed as the field progresses.
In summary, these findings demonstrate that lentiviral transduction of small numbers of transplantable stem cells can achieve sustained, systemic in vivo hAAT expression in mice. In addition to potential use as a technique for gene therapy, this approach could also serve as a valuable laboratory tool to study the putative biological role of anti-proteases in mobilizing blood progenitors from their marrow niche as well as their ability to prevent emphysema in mouse models. Further studies, including the evaluation of alternative, nonviral promoters; targeting of resident lung cells; evaluation of safety concerns; and the use of regulatable lentiviral vectors, need to be performed before experiments in human subjects can be considered.