Carol H. Miao

Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells.

Transplantation of genetically modified hematopoietic stem cells (HSCs) is a promising therapeutic strategy for genetic diseases, HIV, and cancer. However, a barrier for clinical HSC gene therapy is the limited efficiency of gene delivery via lentiviral vectors (LVs) into HSCs. We show here that rapamycin, an allosteric inhibitor of the mammalian target of rapamycin complexes, facilitates highly efficient lentiviral transduction of mouse and human HSCs and dramatically enhances marking frequency in long-term engrafting cells in mice. Mechanistically, rapamycin enhanced postbinding endocytic events, leading to increased levels of LV cytoplasmic entry, reverse transcription, and genomic integration. Despite increasing LV copy number, rapamycin did not significantly alter LV integration site profile or chromosomal distribution in mouse HSCs. Rapamycin also enhanced in situ transduction of mouse HSCs via direct intraosseous infusion. Collectively, rapamycin strongly augments LV transduction of HSCs in vitro and in vivo and may prove useful for therapeutic gene delivery.

Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4+ and CD8+ T cells, whereas increased transforming growth factor-beta levels in plasma and the frequency of both CD4+CD25+FoxP3+ and CD4+CD25-Foxp3+ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4+CD25+ cells did not abrogate tolerance induction, adoptive transfer of CD4+ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3-treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-beta levels and the generation of adaptive FVIII-specific CD4+Foxp3+ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

Progress toward inducing immunologic tolerance to factor VIII

A major problem in treating hemophilia A patients with therapeutic factor VIII (FVIII) is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies. These antibodies block (inhibit) the procoagulant function of FVIII and thus are termed inhibitors. The currently accepted clinical method to attempt to eliminate inhibitors is immune tolerance induction (ITI) via a protocol requiring intensive FVIII treatment until inhibitor titers drop. Although often successful, ITI is extremely costly and is less likely to succeed in patients with high-titer inhibitors. During the past decade, significant progress has been made in clarifying mechanisms of allo- and autoimmune responses to FVIII and in suppression of these responses. Animal model studies are suggesting novel, less costly methods to induce tolerance to FVIII. Complementary studies of anti-FVIII T-cell responses using blood samples from human donors are identifying immunodominant T-cell epitopes in FVIII and possible targets for tolerogenic efforts. Mechanistic experiments using human T-cell clones and lines are providing a clinically relevant counterpoint to the animal model studies. This review highlights recent progress toward the related goals of lowering the incidence of anti-FVIII immune responses and promoting durable, functional immune tolerance to FVIII in patients with an existing inhibitor.

Immunomodulation for inhibitors in hemophilia A: the important role of Treg cells.

Approximately 25–30% of the hemophilia A patients develop inhibitory antibodies against Factor VIII (FVIII) following protein-replacement therapy. This problem is also thought to occur following gene-replacement therapy. Recently, many approaches have been investigated to modulate FVIII-specific immune responses in either protein-replacement or gene therapy hemophilia A mouse models. Several promising protocols have been demonstrated to successfully prevent or modulate the formation of anti-FVIII antibodies, including methods to manipulate antigen presentation, development of less immunogenic FVIII proteins, or formulations or gene therapy protocols to evade immune responses, as well as immunomodulation strategies to target either T- and/or B-cell responses. Most of these successful protocols involve the induction of activated Treg cells to create a regulatory immune environment during tolerance induction. Innovative strategies to overcome pre-existing anti-FVIII immune responses and induce long-term tolerance in primed subjects still need to be developed.

Engineering protein-secreting plasma cells by homology-directed repair in primary human B cells

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

Cell therapy for hemophilia

In this issue of Blood , Follenzi and colleagues demonstrate that transplantation of the bone marrow cells into hemophilia A mice partially restored factor VIII (FVIII) production and protected hemophilia A mice from bleeding challenge. 1 Surprisingly, in recipient hemophilia A mice, the donor BM-derived hepatocytes or endothelial cells were rare; the donor-derived mononuclear cells and mesenchymal stromal cells (MSCs) contributed to major factor VIII gene expression and activity.

434. Adoptive Treg Cell Therapy Using Factor VIII-Specific CAR Regulatory T Cells Regulates Anti-Factor VIII Immune Responses in Hemophilia A Mice

The immune response to factor VIII (FVIII; F8 in constructs) protein limits the effectiveness of treatments for hemophilia A (HemA) patients. Our previous studies demonstrated that regulatory T cells (Tregs) play a pivotal role in modulation of anti-FVIII immune responses. In particular, adoptive transfer of Tregs isolated from FVIII-primed HemA/Foxp3 mice attenuated anti-FVIII immune responses induced by gene transfer of FVIII plasmid in HemA mice. For developing adoptive Treg therapy, we successfully expanded FVIII-sensitized polyclonal Tregs using an FVIII-specific expansion protocol in vitro and showed that these cells had increased FVIII-specific suppressive activity compared with nonspecific Tregs. However, FVIII-specific Tregs in the polyclonal population are still in very small numbers. In this study, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII antibody-derived variable region (scFv) linked to signaling and costimulatory moieties of immune receptors (third generation CAR) and fused with a murine Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro suppressive assay showed that transduced CD4+T cells had significantly higher FVIII-specific suppressive activity than untransduced cells towards FVIII-specific CD4+ effector T cells (Teffs). In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice. One day after cell transfer, the treated mice were challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies. We have also prepared a LV incorporating only the F8CAR region (F8CAR-LV). CD4+CD25− and CD4+CD25+ cells isolated from HemA mice were transduced with F8CAR-LV to generate FVIII-specific Teffs and Tregs, respectively. In vitro FVIII-specific suppressive assays using CD4+ Teffs from FVIII-primed HemA mice or F8CAR-LV transduced Teffs as responder cells are performed to compare the suppressive function of F8CAR-LV transduced CD4+CD25+ cells and F8CAR-Foxp3-LV transduced CD4+ cells. CD4+CD25+ cells isolated from FVIII-primed HemA mice and untransduced CD4+CD25+ and CD4+ cells from HemA mice are used as control cells. These experiments compare the extent of suppression towards specific F8CAR Teffs and polyclonal FVIII-specific Teffs as well as the potency of suppressive function between two differently engineered F8CAR Tregs. Finally, adoptive transfer experiments into HemA mice using the transduced and control cell populations are performed to evaluate their in vivo function to protect the HemA mice from anti-FVIII antibody production. We anticipate that compared with nonspecific and polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.

5. Development of Enhancing Intraosseous Delivery Efficiency of LV-Factor VIII Variants in Platelets of Hemophilia A Mice

Introduction: Our previous studies demonstrated that human factor VIII (FVIII) specifically expressed in megakaryocytes and then stored in platelets of Hemophilia A (HemA) mice can partially correct their phenotype over 5 months in mice with or without pre-existing inhibitors. This was achieved by intraosseous (IO) delivery of lentiviral vectors (LVs) carrying a transgene encoding human FVIII variant (BDDhFVIII/N6; abbreviated as F8) driven by a megakaryocyte-specific promoter (Gp1bα) without preconditioning as required in ex vivo gene therapy. Methods: In this study, we aimed at enhancing transgene expression by two strategies. One was to enhance LV transduction efficiency by suppressing the innate and adaptive immune responses against LVs and LV-transduced cells using pharmacological agents. The other was to improve FVIII gene expression by incorporating a new human FVIII variant, F8X10K12 (a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain; a kind gift from Dr. Weidong Xiao). Results: First, the immune competent C57BL6 mice were pretreated with both dexamethasone (Dex) (IP, 5 mg/kg at -24h, -4h, 4h and 24h) and anti-CD8α monoclonal antibody (mAb) (IP, 4 mg/kg on day -1, 4, 11, 16 and 21), or Dex only. IO infusion of GFP-LVs (1.1×108 i.f.u./mouse) driven by a ubiquitous MND promoter was performed on day 0. On day 7, Dex only and combination drugs + LVs treated mice (n=3) produced higher numbers of GFP+ total bone marrow cells (17.7±3.5% and 11.8±2.1% vs 6.9±3.1%, P=0.0001 and 0.005) and GFP+Lineage-Sca1+cKit+ HSCs (55.5±3.1% and 48.3±6.1% vs 44.4±17.2%, P=0.1 and 0.31) compared with LV-only treated mice (n=3). Most importantly, in the long term, higher numbers of GFP+ cells (2.4±0.4% vs 0.5±0.1%, P<0.001) in the total bone marrow and GFP+HSCs (10.7±3.3% vs 2.6±0.6%, P<0.001) were observed in combination drugs + LVs treated mice (n=3) compared with LV-only treated mice on day 160 after LV infusion (n=3), which was confirmed by higher LV copy number in bone marrow cells of drugs + LVs treated mice. Second, we tested the FVIII expression levels from two human FVIII variants in HemA mice by hydrodynamic injection of plasmids driven by a human elongation factor-1 promoter (pEF1α-F8X10K12 or pEF1α-F8, 50 µg/mouse, n=8), respectively. Compared with F8, F8X10K12 produced a 25-fold increase (147±27% vs 3,734±477%) in the clotting activity determined by an aPTT assay on day 4 post injection. Then two LVs containing F8X10K12 or F8 transgene driven by EF1α promoter (E-F8X10K12-LV or E-F8-LV) were constructed and used to transduce 293T cells, respectively. Flow cytometry data showed that E-F8X10K12-LV produced a significant increase of hFVIII+293T cells (77.8% vs 15%) and MFI (795 vs 541) compared to E-F8-LV at the same doses. These results indicated that F8X10K12 may further enhance FVIII gene expression in platelets for more effective therapy. LVs containing F8X10K12 or F8 transgene driven by Gp1bα promoter (G-F8X10K12-LV or G-F8-LV) were subsequently generated and were intraosseously delivered into HemA mice to test the FVIII efficiency in platelets by ELISA, thrombin generation assay and tail clipping. Conclusion: We found that administration of Dex that efficiently inhibited initial innate immune responses to LVs in vivo combined with anti-CD8α mAb that depleted subsequent cytotoxic CD8+ T cells improved the transduction efficiency of LVs and persistence of transduced cells, leading to over 10% GFP+HSCs in treated mice up to 160 days. In addition, a new FVIII variant, F8X10K12, can significantly enhance FVIII expression in mice following hydrodynamic injection of plasmids and in LV-transduced cells. Taken together, IO infusion of G-F8X10K12-LV into HemA mice pretreated with Dex and antiCD8α mAb can be used to further enhance and prolong transgene efficiency in platelets for effective correction of hemophilia A.

441. IL2 Plasmid Treated Hemophilia A Mice Show an Increase in Regulatory T Cell Population and Initial Tolerance to FVIII

Hemophilia A is an inherited X chromosomal linked recessive disease. Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. This inability to clot can result in major blood loss and potential death. The current treatment for patients with hemophilia A is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect. In order to overcome this potential immune response patients undergo a high dose regimen of FVIII to induce tolerance. However, a third of patients still develop an immune response.Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Both of these cell types bind to interleukin 2 (IL2), a cytokine which promotes the differentiation, expansion and activation of these cell populations. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII plasmid.Hemophilia A mice were hydrodynamically injected with either 2 µg or 5 µg IL2 plasmid and 50 µg FVIII plasmid, either sequentially (1 week apart) or simultaneously. Both plasmids were driven by a liver-specific promoter (hAAT-HCR). Cell staining data showed a marked increase in Treg cell population and activation, demonstrating that a small amount of IL2 plasmid incorporated into liver cells is enough to dramatically increase the Treg population. A larger increase of Teff cells were observed in mice treated with 5 µg IL2 plasmid than in mice treated with 2 µg IL2 plasmid. Importantly, Treg/Teff ratio was significantly increased in the 2 µg IL2 plasmid group in the first 3 weeks and maintained at increased levels over several weeks afterwards. ELISA showed an initial higher level of IL2 production; IL2 expression dropped and maintained at low levels after one week. On Day 28-post FVIII plasmid injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 plasmid showed no inhibitor development with persistent FVIII gene expression. The treated mice are monitored to determine the potential long term tolerance effects induced by low dose IL2 plasmid. Using gene therapy to slightly increase the amount of a cytokine in patients could provide a better treatment option than existing drug regimens. FVIII protein is also extremely expensive, especially in the amounts needed for treating hemophilia A patients. Combined gene transfer of FVIII and IL2 plasmids has the potential to produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, therefore reducing the amount of expensive reagents needed, the number of doctor visits required, and the overall cost, potential morbidity and stress to the patient.