Moanaro Biswas

Clinical development of gene therapy: results and lessons from recent successes

Therapeutic gene transfer holds the promise of providing lasting therapies and even cures for diseases that were previously untreatable or for which only temporary or suboptimal treatments were available. For some time, clinical gene therapy was characterized by some impressive but rare examples of successes and also several setbacks. However, effective and long-lasting treatments are now being reported from gene therapy trials at an increasing pace. Positive outcomes have been documented for a wide range of genetic diseases (including hematological, immunological, ocular, and neurodegenerative and metabolic disorders) and several types of cancer. Examples include restoration of vision in blind patients, eradication of blood cancers for which all other treatments had failed, correction of hemoglobinopathies and coagulation factor deficiencies, and restoration of the immune system in children born with primary immune deficiency. To date, about 2,000 clinical trials for various diseases have occurred or are in progress, and many more are in the pipeline. Multiple clinical studies reported successful treatments of pediatric patients. Design of gene therapy vectors and their clinical development are advancing rapidly. This article reviews some of the major successes in clinical gene therapy of recent years.

Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell–dependent induction of CD4+CD25+FoxP3+ Treg

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg) are critical elements for maintaining immune tolerance, for instance to exogenous antigens that are introduced during therapeutic interventions such as cell/organ transplant or gene/protein replacement therapy. Coadministration of antigen with rapamycin simultaneously promotes deletion of conventional CD4(+) T cells and induction of Treg. Here, we report that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin. This occurs via selective expansion of plasmacytoid dendritic cells (pDCs), which further augments the number of Treg. Whereas in conventional DCs, rapamycin effectively blocks mammalian target of rapamycin (mTOR) 1 signaling induced by Flt3L, increased mTOR1 activity renders pDCs more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDCs. This concept is supported by the finding that Treg induction is abrogated upon pDC depletion. The combination with pDCs and rapamycin is requisite for Flt3L/antigen-induced Treg induction because Flt3L/antigen by itself fails to induce Treg. As co-administering Flt3L, rapamycin, and antigen blocked CD8(+) T-cell and antibody responses in models of gene and protein therapy, we conclude that the differential effect of rapamycin on DC subsets can be exploited for improved tolerance induction.

Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft versus host disease in bone marrow transplantation. Here, we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e., hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express green fluorescent protein–marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 100-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within 2 weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies.

Plasmacytoid and conventional dendritic cells cooperate in crosspriming AAV capsid-specific CD8+ T cells

Adeno-associated virus (AAV) is a replication-deficient parvovirus that is extensively used as a gene therapy vector. CD8+ T-cell responses against the AAV capsid protein can, however, affect therapeutic efficacy. Little is known about the in vivo mechanism that leads to the crosspriming of CD8+ T cells against the input viral capsid antigen. In this study, we report that the Toll-like receptor 9 (TLR9)-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating this response. By contrast, the absence of TLR2, STING, or the addition of TLR4 agonist has no effect. Surprisingly, both conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are required for the crosspriming of capsid-specific CD8+ T cells, whereas other antigen-presenting cells are not involved. TLR9 signaling is specifically essential in pDCs but not in cDCs, indicating that sensing of the viral genome by pDCs activates cDCs in trans to cross-present capsid antigen during CD8+ T-cell activation. Cross-presentation and crosspriming depend not only on TLR9, but also on interferon type I signaling, and both mechanisms can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. Thus, these outcomes directly point to therapeutic interventions and demonstrate that innate immune blockade can eliminate unwanted immune responses in gene therapy.

Gene Therapy With Regulatory T Cells: A Beneficial Alliance

Gene therapy aims to replace a defective or a deficient protein at therapeutic or curative levels. Improved vector designs have enhanced safety, efficacy, and delivery, with potential for lasting treatment. However, innate and adaptive immune responses to the viral vector and transgene product remain obstacles to the establishment of therapeutic efficacy. It is widely accepted that endogenous regulatory T cells (Tregs) are critical for tolerance induction to the transgene product and in some cases the viral vector. There are two basic strategies to harness the suppressive ability of Tregs: in vivo induction of adaptive Tregs specific to the introduced gene product and concurrent administration of autologous, ex vivo expanded Tregs. The latter may be polyclonal or engineered to direct specificity to the therapeutic antigen. Recent clinical trials have advanced adoptive immunotherapy with Tregs for the treatment of autoimmune disease and in patients receiving cell transplants. Here, we highlight the potential benefit of combining gene therapy with Treg adoptive transfer to achieve a sustained transgene expression. Furthermore, techniques to engineer antigen-specific Treg cell populations, either through reprogramming conventional CD4+ T cells or transferring T cell receptors with known specificity into polyclonal Tregs, are promising in preclinical studies. Thus, based upon these observations and the successful use of chimeric (IgG-based) antigen receptors (CARs) in antigen-specific effector T cells, different types of CAR-Tregs could be added to the repertoire of inhibitory modalities to suppress immune responses to therapeutic cargos of gene therapy vectors. The diverse approaches to harness the ability of Tregs to suppress unwanted immune responses to gene therapy and their perspectives are reviewed in this article.

Innovative Approaches for Immune Tolerance to Factor VIII in the Treatment of Hemophilia A

Hemophilia A (coagulation factor VIII deficiency) is a debilitating genetic disorder that is primarily treated with intravenous replacement therapy. Despite a variety of factor VIII protein formulations available, the risk of developing anti-dug antibodies (“inhibitors”) remains. Overall, 20–30% of patients with severe disease develop inhibitors. Current clinical immune tolerance induction protocols to eliminate inhibitors are not effective in all patients, and there are no prophylactic protocols to prevent the immune response. New experimental therapies, such as gene and cell therapies, show promising results in pre-clinical studies in animal models of hemophilia. Examples include hepatic gene transfer with viral vectors, genetically engineered regulatory T cells (Treg), in vivo Treg induction using immune modulatory drugs, and maternal antigen transfer. Furthermore, an oral tolerance protocol is being developed based on transgenic lettuce plants, which suppressed inhibitor formation in hemophilic mice and dogs. Hopefully, some of these innovative approaches will reduce the risk of and/or more effectively eliminate inhibitor formation in future treatment of hemophilia A.

Immune Modulatory Cell Therapy for Hemophilia B Based on CD20-Targeted Lentiviral Gene Transfer to Primary B Cells

Gene-modified B cells expressing immunoglobulin G (IgG) fusion proteins have been shown to induce tolerance in several autoimmune and other disease models. However, lack of a vector suitable for gene transfer to human B cells has been an obstacle for translation of this approach. To overcome this hurdle, we developed an IgG-human factor IX (hFIX) lentiviral fusion construct that was targeted to specifically transduce cells expressing human CD20 (hCD20). Receptor-specific retargeting by mutating envelope glycoproteins of measles virus (MV)-lentiviral vector (LV) and addition of a single-chain variable fragment specific for hCD20 resulted in gene delivery into primary human and transgenic hCD20 mouse B cells with high specificity. Notably, this protocol neither required nor induced activation of the B cells, as confirmed by minimal activation of inflammatory cytokines. Using this strategy, we were able to demonstrate induction of humoral tolerance, resulting in suppression of antibody formation against hFIX in a mouse model of hemophilia B (HB). In conclusion, transduction of receptor-specific retargeted LV into resting B cells is a promising method to develop B cell therapies for antigen-specific tolerance induction in human disease.

Regulatory T cells and TLR9 activation shape antibody formation to a secreted transgene product in AAV muscle gene transfer

Adeno-associated viral (AAV) gene delivery to skeletal muscle is being explored for systemic delivery of therapeutic proteins. To better understand the signals that govern antibody formation against secreted transgene products in this approach, we administered an intramuscular dose of AAV1 vector expressing human coagulation factor IX (hFIX), which does not cause antibody formation against hFIX in C57BL/6 mice. Interestingly, co-administration of a TLR9 agonist (CpG-deoxyoligonucleotide, ODN) but not of lipopolysaccharide, caused a transient anti-hFIX response. ODN activated monocyte-derived dendritic cells and enhanced T follicular helper cell responses. While depletion of regulatory T cells (Tregs) also caused an antibody response, TLR9 activation combined with Treg depletion instead resulted in prolonged CD8+ T cell infiltration of transduced muscle. Thus, Tregs modulate the response to the TLR9 agonist. Further, Treg re-population eventually resolved humoral and cellular immune responses. Therefore, specific modes of TLR9 activation and Tregs orchestrate antibody formation in muscle gene transfer.

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Summary Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+ CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

293. Optimal In Vivo Treg Induction and Suppression of Immune Responses By Synergistic Use of Rapamycin and FLT3 Ligand

Immune-mediated rejection of therapeutic proteins is a major hurdle for gene and protein replacement therapies for genetic disease. However, optimal induction and expansion of CD4+CD25+FoxP3+ regulatory T cells (Treg) counteract effector responses. We have previously shown that co-administration of antigen with rapamycin simultaneously promotes deletion of conventional CD4+ T cells and induction of Treg, so that we were able to induce tolerance to factors VIII and IX in treatment of the X-linked bleeding disorders hemophilia A and B. Here, we found in DO11.10 Rag−/− mice (homozygous for ovalbumin-peptide specific TCR, deficient in endogenous mature B, T or Treg cells) that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin via selective expansion of plasmacytoid dendritic cells (pDC), resulting in substantially improved induction of OVA-specific Treg. This regimen, consisting of co-administration of rapamycin/Flt3L/OVA 3x/week for 1 month, also prevented OVA-specific CD8+ T cell responses in immune competent mice after intrasmuscular OVA gene transfer with an AAV1 vector. Flt3L signaling is critical to the generation and steady state expansion of both the conventional (CD11c+, CD8+CD11c+) and plasmacytoid (CD11cmid-loPDCA-1+) subsets of DC, which indirectly expands naturally occurring Treg. Interestingly, Flt3L-induced signaling in DC and DC precursors occurs through the mTOR pathway and can therefore be blocked by rapamycin, posing the question of why we are able to use these drugs synergistically for Treg induction. We find that limited doses of rapamycin selectively block Flt3L-induced expansion of conventional DC (cDC) but not pDC. Induced mTOR signaling is effectively blocked in cDC, while pDC have increased mTOR activity and are thus more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDC. During Flt3L/rapamycin/antigen co-administration, Treg induction is abrogated upon pDC depletion, thus confirming that Treg induction is pDC dependent. Similarly, Flt3L/antigen by itself fails to induce Treg, indicating that both pDC and rapamycin are required for Treg induction in this regimen.Finally, we are able to demonstrate in a therapeutic model for FVIII protein replacement therapy that combined Flt3L/rapamycin/FVIII treatment is superior in prevention of inhibitory antibody responses in hemophilia A mice (BALB/c F8e16−/−) when compared to rapamycin/FVIII only. In contrast, Flt3L/antigen administration in the absence of rapamycin, while enhancing activation induced cell death (AICD) of conventional CD4+ T cells, fails to induce Treg and also fails to induce tolerance to OVA or to FVIII. Thus, the differential effect of rapamycin on DC subsets can be exploited to improve tolerance induction via enhanced Treg induction, creating new opportunities for development of immune tolerance therapies with wide applicability.