Patients with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have extremely poor survival when treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance of cure for these patients, but attaining clinical remission in order to undergo HSCT remains the biggest therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has been used with great success in relapsed/refractory B-ALL patients. The same approach, however, is difficult in targeting T-cell disease given the lack of a T-lymphoblast specific surface antigen. The use of natural killer (NK) cells as the effector cell provides a potential solution. We are testing two different CAR structures directed against CD5, a pan T-cell marker; one consisting of a more traditional immunoglobulin (Ig) based single chain variable fragment (scFv), while the other comprising of a variable lymphocyte receptor (VLR) as the antigen recognition domain. VLRs are single chain crescent shaped proteins that represent the functional unit of the adaptive immune system in jawless vertebrates. Our previous work has shown that a VLR can be used as an alternative for CAR-based antigen recognition. Here, we directly compare a VLR to its scFv counterpart in a CAR setting. The single chain structure of a VLR allows for the rapid creation of novel CARs, compared to the corresponding use of a scFv-CAR, in which the variable heavy and light sequences need further engineering for adapting to CAR technologies. Thus, our hypothesis was that natural killer cells engineered to express an anti-CD5 CAR can specifically be used to target T-cell malignancies, with the VLR-CAR being equal or superior to scFv-based cassettes. We constructed two second generation CARs using the VLR or scFv sequence targeting CD5 as our antigen recognition domain. Both CD5-CAR constructs showed similar activity when tested in CD5-positive Jurkat cells. For our cytotoxicity studies, we used the IL-2 dependent natural killer cell line NK-92. To generate uniform populations of CAR-expressing NK cells we transduced NK-92 cells with a dual lentiviral construct expressing the CD5-CAR and green fluorescent protein (GFP), and then selected for the positively transduced cells using flow sorting for GFP. In vitro cytotoxicity studies showed significantly increased killing using the CD5-CAR expressing NK-92 cells compared to unmodified NK-92 cells (p<0.05), for both VLR and scFv modified cells. We then tested the two CD5-CARs in a T-cell leukemia mouse model. NOD scid gamma (NSG) mice were intravenously injected with luciferase expressing Jurkat leukemia cells to establish the T-cell leukemia xenograft model. Four doses of NK-92 cells were injected over a two week period starting on day 7 post-tumor injection. Bioluminescence imaging was used to monitor tumor burden. The CD5-scFv-CAR NK-92 treated mice group had a significant survival advantage over the other groups (p<0.01). Interestingly, the CD5-VLR-CAR NK-92 treated mice did not have a significant advantage over naïve NK-92 or saline treated groups. Given the superior in vivo cytotoxicity demonstrated in our studies, we plan to use the CD5-scFV-CAR going forward as we transition to primary cells. Overall, these studies provide the foundation to move these studies into preclinical testing as a treatment for relapsed T-ALL.

Relapsed T-cell malignancies have poor outcomes when treated with chemotherapy, but survival after allogeneic bone marrow transplantation (BMT) approaches 50%. A limitation to BMT is the difficulty of achieving remission prior to transplant. Chimeric antigen receptor (CAR) T-cell therapy has shown successes in B-cell malignancies. This approach is difficult to adapt for the treatment of T-cell disease due to lack of a T-lymphoblast specific antigen and the fratricide of CAR T cells that occurs with T-cell antigen targeting. To circumvent this problem two approaches were investigated. First, a natural killer (NK) cell line, which does not express CD5, was used for CAR expression. Second, CRISPR-Cas9 genome editing technology was used to knockout CD5 expression in CD5-positive Jurkat T cells and in primary T cells, allowing for the use of CD5-negative T cells for CAR expression. Two structurally distinct anti-CD5 sequences were also tested, i) a traditional immunoglobulin-based single chain variable fragment (scFv) and ii) a lamprey-derived variable lymphocyte receptor (VLR), which we previously showed can be used for CAR-based recognition. Our results show i) both CARs yield comparable T-cell activation and NK cell-based cytotoxicity when targeting CD5-positive cells, ii) CD5-edited CAR-modified Jurkat T cells have reduced self-activation compared to that of CD5-positive CAR-modified T cells, iii) CD5-edited CAR-modified Jurkat T cells have increased activation in the presence of CD5-positive target cells compared to that of CD5-positive CAR-modified T cells, and iv) although modest effects were seen, a mouse model using the CAR-expressing NK cell line showed the scFv-CAR was superior to the VLR-CAR in delaying disease progression.

Remission reinduction poses a great challenge in treating relapsed T-cell malignancies. These patients have poor outcomes when treated with chemotherapy alone. Treatment of relapsed B-cell malignancies using chimeric antigen receptor (CAR) T-cell therapy has had great success. However, the same approach will be difficult to apply to the treatment of T-cell malignancies due to the lack of T-lymphoblast specific antigens. Therefore, T cells modified with anti-T cell CARs target themselves, potentially resulting in fratricide of CAR T cells and killing of normal T cells. We have been investigating mechanistic approaches to evade fratricide-based limitations of CAR therapy for T-cell leukemias. Since the majority of T-cell leukemias express CD5, we hypothesized that if CD5 expression was knocked down/out in effector T cells, the issue of fratricide would be eliminated. We used CRISPR/Cas9 genome editing to knock out CD5 expression in CD5-positive T cells, resulting in CD5-negative T cells that were used as CAR-expressing effector cells. In order to target CD5, CARs were generated using two structurally different antigen recognition sequences - a traditional immunoglobulin-based single chain variable fragment (scFv) and a lamprey-derived variable lymphocyte receptor (VLR). We have previously shown this VLR can be used as an alternative for CAR-based antigen recognition. Here, we compare non-CRISPR T cells to CD5-edited T cells when modified with either a CD5-VLR-CAR or CD5-scFv-CAR. Our in vitro studies show both CARs yield comparable T-cell activation in non-CRISPR T cells and that CD5-edited CAR-modified T cells have a substantial reduction in self-activation. Furthermore, CD5-edited CAR-modified cells become activated in the presence of CD5-positive target cells to a greater degree than do non-CRISPR CAR-modified T cells (p<0.05 at all effector:target ratios of 2:1, 1:1 and 1:5). We, and others, have shown a decrease in cell surface CD5 protein expression following lentiviral gene transfer of a CD5-CAR transgene cassette in non-CD5 edited T cells using both VLR and scFv-based vectors. This downregulation has been speculated to be due to the interaction of the cell surface CAR with the target antigen. We also show that CD5-CAR expression in CD5-edited CAR-modified T cells is increased compared to CD5-CAR expression in non-CRISPR CAR-modified T cells (see Figure). Therefore, the interaction of the CAR with the cell surface antigen not only decreases antigen levels, but CAR levels as well. The decreased fratricide, increased CAR expression and activation of CD5-edited CAR-modified T cells in the presence of target cells should lead to more potent and durable anti-leukemia effects. Overall, our preclinical data show an advantage to using CD5-edited effector T cells when utilizing CAR-T cell therapy to target difficult to treat relapsed T-cell malignancies.

Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naïve cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.

Relapsed T-cell leukemia has a poor prognosis. Chimeric antigen receptor (CAR) therapy could be an effective treatment modality, although targeting T-cell disease without a T-lymphoblast specific antigen is difficult. The use of natural killer (NK) cells as the effector cell and CD5 as the target antigen provides a potential solution. While traditionally CARs use an immunoglobulin (Ig) based single chain variable fragment (scFV) to target tumor cells, we designed a novel CAR structure using a variable lymphocyte receptor (VLR) as our antigen recognition sequence. VLRs represent the functional unit of the adaptive immune system in jawless vertebrates (lamprey and hagfish) and are analogous but not homologous to immunoglobulins. VLRs have a fundamentally different structure and geometry compared to Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures that allows for rapid creation of CAR cassettes. In the current study our objective was to develop a VLR-CAR-NK cell against T-cell leukemia using an anti-CD5 VLR sequence. We constructed a second generation CAR using a VLR sequence targeting CD5 as our antigen recognition sequence. The CAR structure consisted of the anti-CD5 VLR sequence, the CD28 co-stimulatory transmembrane domain, and the intracellular CD3ζ signaling domain. Using this construct, high titer CD5 VLR CAR self-inactivating lentivirus was produced at titers >10^8 TU/ml. To test the functionality of the CAR construct, CD5 expressing Jurkat cells were transduced with various doses of lentivirus, and activation was measured by expression of CD69. For cytotoxicity studies, NK-92 cells were used as the effector cell with CCRF-CEM cells being the target cell. Cytotoxicity was measured at different effector: target ratios using a flow cytometry based assay. Activation positively correlated with lentiviral copy numbers and CAR expression in Jurkat cells. Both copy number and activation decreased over time, which was anticipated as we show that highly activated cells had a growth disadvantage. NK-92 cells expressing the CD5 VLR CAR showed approximately two fold increase in cytotoxicity towards CCRF-CEM cells when compared with naïve NK-92 cells; however, low transduction efficiency and copy numbers (<0.5) were problematic, similar to previous data showing that NK-92 cells are resistant to lentiviral transduction. In order to improve upon these results, we created a bicistrionic vector co-expressing GFP and the CD5-VLR-CAR. Jurkat cells genetically modified to express the new construct showed a direct correlation between GFP expression and activation, thus confirming dual expression and function of both proteins. Transduced NK-92 cells were sorted and expanded. These cells expand robustly, demonstrate CAR expression and have the functional characteristics required for targeting T-cell disease. Our studies show the utility of VLR-derived CARs and provide the foundation to progress these studies into preclinical testing as a treatment for relapsed T-cell leukemia.

Chimeric antigen receptors (CARs) are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs). VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig)-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

Although chimeric antigen receptors (CARs) are yielding promising clinical results, the technology remains limited by the availability of conjugate cancer cell target antigens. As a means to increase both the identification of cancer cell target antigens and simultaneously generate unique single chain binding domain structures we utilized the immune system of jawless vertebrates. Sea lamprey possess an adaptive immune system primarily characterized by variable lymphocyte receptors (VLRs) as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins (Ig). VLRs have a fundamentally different structure and geometry than Ig-based antibodies while still demonstrating high degrees of specificity and avidity. Additionally, VLRs exist naturally as single chain structures with their variable region consisting of multiple assembled repeating sequences termed leucine rich repeats. These repeats can be directly inserted onto a CAR scaffold by genetic engineering. To test this platform technology, a yeast display method previously described (Tasumi et al., PNAS 2009; Xu et al., Humana press 2011) was used as a means of assaying and selecting appropriate VLRs. VLRs meeting the set criteria are sequenced and cloned into a lentiviral vector (LV) CAR transgene cassette plasmid. We constructed a CAR containing a well characterized VLR specific for the B cell receptor of a murine B cell leukemia (BCL) cell line. The CAR design incorporates the anti-BCL-VLR, Myc tag, CD28 transmembrane domain, and the intracellular CD3ζ signaling domain. SIN VLR-CAR LV was produced at high titer (~1x108) and used to transduce Jurkat cells. Transduced Jurkat cells showed successful CAR protein expression confirmed via Western Blot as well as persistent surface CAR expression for over 2 months with cell viability remaining over 85%. To determine whether the VLR was capable of signaling through the CAR, transduced Jurkat cells were incubated with the BCL cell line expressing the target B cell receptor. Using this assay, we demonstrated potent T cell activation via the VLR-CAR. CAR expressing T-cells demonstrated activation in ~80% of the cells. Furthermore, NK-92 cells expressing the VLR-CAR demonstrated an ability to recognize and kill BCL cells at target to effector ratios as low as 1:1, with 30% target cell killing. Little to no killing was observed with a control B cell line. Additionally, we created a CAR using a published VLR sequence targeting the human surface antigen CD5 (Yu et al., J. Immunol Methods, 2012). CD5 is primarily a T-cell marker, thus an anti-CD5-VLRCAR could be potentially used to target T-cell malignancies. Jurkat cells were transduced with high titer SIN VLR-CAR LV at various MOIs. Since Jurkat cells express CD5 on their surface, we tested the different transduced groups for self-activation at several time points. The corresponding transduced copy number was determined using quantitative PCR. The degree of activation directly correlated with the LV copy number, with highest activation being seen when the MOI approach 50, with >60% highly activated cells. No activation was observed in the GFP control groups. Additionally, we engineered three different CAR constructs consisting of two CD5 VLRs connected by either a helical linker with a 180° rotation, a helical linker with a 360° rotation, or a non-rigid linker allowing for flexibility among the two VLRs. Transduced Jurkat cells expressing the double CD5-VLR CARs indicated, via flow cytometry, that linking two VLRs does not provide any benefit in terms of activation compared to the single CD5-VLR-CAR. Current testing includes the use of NK-92 cells expressing the anti-CD5-VLR-CAR against T cell leukemia cell lines. Collectively, these results show that VLRs can be used to increase the repertoire of CAR binding motifs. Furthermore, these data suggest that VLRs provide both a unique and effective method for activating CAR-T cells and can expand the number and variety of antigens that may be targeted.

Background: Recent clinical trials have demonstrated the efficacy and safety of gene therapy utilizing HIV-derived lentiviral vectors (LVs) for blood disorders. However, the LV requirements and clinical ex vivo cell transduction protocols used in these studies exposes the limitations of the technology and beckons the need for improved LV manufacturing and clinical transduction efficiency. Many methods have been devised to enhance efficiency, although none have circumvented the exorbitant amounts of virus required to achieve therapeutic HSC transduction. Furthermore, prolonged ex vivo cell culture is necessary to achieve sufficient transduction despite exposure to toxic byproducts of LV production. To that end, we developed a novel, scalable microfluidic for clinical LV transduction that leverages mass transfer principles to significantly reduce the amount of LV required to achieve therapeutic levels of gene transfer and transduction time by more efficiently exposing cells to virus.

Cell culture in microfluidic systems has primarily been conducted in devices comprised of polydimethylsiloxane (PDMS) or other elastomers. As polystyrene (PS) is the most characterized and commonly used substrate material for cell culture, microfluidic cell culture would ideally be conducted in PS-based microsystems that also enable tight control of perfusion and hydrodynamic conditions, which are especially important for culture of vascular cell types. Here, we report a simple method to prototype perfusable PS microfluidics for endothelial cell culture under flow that can be fabricated using standard lithography and wet laboratory equipment to enable stable perfusion at shear stresses up to 300 dyn/cm(2) and pumping pressures up to 26 kPa for at least 100 h. This technique can also be extended to fabricate perfusable hybrid PS-PDMS microfluidics of which one application is for increased efficiency of viral transduction in non-adherent suspension cells by leveraging the high surface area to volume ratio of microfluidics and adhesion molecules that are optimized for PS substrates. These biologically compatible microfluidic devices can be made more accessible to biological-based laboratories through the outsourcing of lithography to various available microfluidic foundries.

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloiderythroid- leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Here, we tested a novel, nonablative transplantation therapy in two pediatric HA animals. Paternal mesenchymal stem cells (MSC) were transduced with a porcine FVIII-encoding lentivector and transplanted via the intraperitoneal route without preconditioning. At the time of transplantation, these animals had received multiple human FVIII treatments for various spontaneous bleeds and had developed debilitating hemarthroses, which produced severe defects in posture and gait. Transplantation of transduced MSC resolved all existent hemarthroses, and spontaneous bleeds ceased. Damaged joints recovered fully; the animals regained normal posture and gait and resumed normal activity. Despite achieving factor-independence, a sharp rise in pre-existent Bethesda titers occurred following transplantation, decreasing the effectiveness and duration of therapy. Postmortem examination revealed widespread engraftment, with MSC present within the lung, liver, intestine, and thymus, but particularly within joints affected at the time of transplantation, suggesting MSC homed to sites of ongoing injury/inflammation to release FVIII, explaining the dramatic improvement in hemarthrotic joints. In summary, this novel, nonablative MSC transplantation was straightforward, safe, and converted life-threatening, debilitating HA to a moderate phenotype in a large animal model.

Abstract Abstract 249 We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Herein, we tested the ability of a novel, postnatal, non-ablative transplant approach to correct the disease in 2 of the HA animals. Haploidentical (paternal) bone marrow mesenchymal stem cells (MSC) were transduced simultaneously with lentiviral vectors encoding a high expression porcine FVIII (pFVIII) or an eGFP transgene, to facilitate donor cell identification. Upon sedation, MSC were injected into the peritoneal cavity of the young adult animals by ultrasound-guided delivery, without any preconditioning. At the age of 4 months, the time of the first transplantation (Tx), animal #1 (HA#1) had received 32,200U of recombinant hFVIII in 22 treatments for muscular hematomas, rectal bleeding, and chronic, progressive, debilitating hemarthroses which had caused severe defects in posture and gait, rendering him nearly immobile. Low levels of hFVIII inhibitors (4-6) were detected by Bethesda assay at this time point. Tx of 30×10⋀6 transduced MSC resolved all existent hemarthroses, and resulted in a cessation of all spontaneous bleeding events. HA#1's damaged joints recovered fully; he regained normal posture and gait, and resumed normal physical activity. Despite HA#1 becoming and remaining factor-independent, however, a rise in Bethesda titer to ≂f625 was observed. At 5.5 months of age, a 2nd Tx with 120×10⋀6 transduced MSC was performed to test whether higher cell numbers would induce immune tolerance to FVIII; however, the levels of FVIII antibodies remained the same. At 7 months, HA#1 was euthanized after ingesting a sharp rock which caused a massive hematoma of the larynx, reducing his airway by >75%. Tissue analysis demonstrated large numbers of GFP+/FVIII+ MSC present within the synovium of the joints which had hemarthrosis at the time of the 1st Tx, suggesting the transplanted MSC homed to the sites of ongoing injury/inflammation and persisted for over 3 months, releasing FVIII locally within the joint, providing an explanation for the dramatic improvement we observed in this animal's joints. In order to elucidate the mechanism whereby this transplant approach produced such pronounced systemic benefit, we performed PCR and confocal analysis on other tissues. PCR has demonstrated engraftment of transplanted MSC in all tissues thus far analyzed, including liver, lymph nodes, intestine, lung, kidney, omentum, and thymus. Confocal analysis revealed significant levels of engraftment within the lymph nodes, small intestine and the thymus. Analysis of other tissues is ongoing to fully define the sites of engraftment, and understand how these engrafted cells producing FVIII locally are exerting widespread clinical improvement. A 2nd HA animal (HA#2) was transplanted at 5 months of age with 120×10⋀6 transduced paternal MSC. In similarity to HA#1, hemarthroses present at the time of Tx resolved, and he resumed normal activity. However, despite having no detectable inhibitors prior to Tx, HA#2 developed titers of ≂f150 following this procedure. HA#2 is alive, has exhibited no spontaneous bleeds, and has only required FVIII treatment to resolve bleeding resulting from accidental trauma. His plasma is being assayed regularly to determine if the continued production of FVIII by the transplanted MSC will result in tolerance induction and a drop in inhibitor titer. In summary, this novel postnatal, nonmyeloablative MSC Tx successfully converted both animals from a severe, life-threatening phenotype to a moderate phenotype, devoid of spontaneous bleeds. Further mechanistic studies will be required to understand and ultimately overcome the formation of inhibitors resultant from this procedure since, in the absence of inhibitors, this approach could result in even more pronounced clinical improvement. Disclosures: No relevant conflicts of interest to declare.

Background Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response.Methods Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, NM expression and activity, and generation of anti-fVIII immune response.Results We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of NM function; (iii) naive and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII.Conclusions These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products. Copyright (C) 2010 John Wiley & Sons, Ltd.