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Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 T cells including CAR-T products, CAR-T cells, and endogenous T cells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous T cell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8(+) CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.

Top of pageAbstract Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroid- specific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0 |[ndash]| 100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring long-term hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.

Recombinant lentiviral vectors derived from human immunodeficiency virus-1 are promising vectors for gene therapy applications. This is due to their ability to transduce nondividing cells, their genomic stability, their relatively large genomic capacity, as well as their ability to stably maintain long term transgene expression. Recent studies have uncovered that the central polypurine tract (cPPT) plays an important role in increasing lentivirus-mediated gene transfer efficiency. The cPPT participates in lentiviral reverse transcription by initiating the synthesis of a downstream plus strand cDNA. Together with the upstream plus strand initiated at the 3' polypurine tract, it forms a central DNA flap that bears in its center a short plus strand overlap. A possible role in nuclear import enhancement of the viral preintegration complex has been suggested (Zennou, Cell, 2000), but the mechanism of cPPT function is still not fully elucidated. It has also been reported that the incorporation of cPPT in lentiviral vectors can increase particle infectivity (Follenzi, Nature Genetics, 2000). However, a systematic evaluation of the cPPT in complex lentiviral vectors has not been reported to date.

Current attempts to treat hemophilia with stem cell-based gene therapy make use of oncoretroviral or lentiviral vectors with ubiquitous promoters to express human FVIII or FIX. However, studies in mouse models of hemophilia have resulted in low transgene expression (Evans & Morgan, 1998; Kootstra & Verma, 2003; Tiede & Scherr, 2003), only achieving therapeutic expression levels following high copy gene transfer (>5 copies/cell, Bigger and Thermis, 2004). A recent study by Moayeri et al. (Mol. Ther., 2004) demonstrated long-term therapeutic levels of factor VIII expression in immunocompromised hemophilic mice engrafted with pre-selected, oncoretroviral vector transduced hematopoietic stem cells (HSCs). However, non-specific promoters may pose a greater risk of inducing insertional oncogenesis than vectors whose expression is restricted to terminally differentiated progeny, such as maturing erythroblasts.