Hemophilia is a hematological disorder caused by mutations in the X-linked encoding coagulation factor VIII (hemophilia A) or IX (hemophilia B). Hemophilia occurs in 1 in 5,000 births worldwide. In its severe form (<1% coagulation activity), failure of blood clotting causes spontaneous—and potentially life threatening—bleeds into joints and soft tissues. Patients with hemophilia are currently treated with intravenous infusion of factor protein concentrate, which may have to be administered up to 3 times per week to prevent serious internal bleeds. This lifelong treatment is burdensome and expensive. Due to the costs of the protein drugs, treatment is not typically available in underdeveloped nations.
In contrast, gene therapy has the potential to be curative and lasting for many years after a single round of gene transfer, which has now materialized in humans in clinical trials. This was accomplished by hepatic gene transfer using adeno-asscoiated viral (AAV) vectors, an approach that Herzog helped to pioneer.
The Herzog Lab continues to study and refine this approach with four primary goals: Develop vectors that are superior in gene transfer to human hepatocytes; define the mechanism of the immune response to AAV and ultimately prevent B and T cell responses to the vector; achieve immune tolerance to the therapeutic transgene product (FVIII or FIX); and define the mechanism by which immune tolerance induction is achieved.
A seminal finding in the lab was that hepatic gene transfer with AAV vectors can induce immune tolerance to the transgene product by induction of CD4+CD25+FoxP3+ regulatory T cells (Treg), deletion of effector T cells and direct suppression of memory B cells. MHC II presentation to CD4+ T cells occurs primarily in draining lymph nodes of the liver and requires both macrophages and dendritic cells. Induced Treg distribute systemically, resulting in enforcement of immune tolerance in other organs. Hepatic AAV gene transfer can be used not only to prevent but also reverse immune responses, for example to eliminate formation of pathogenic antibodies and anaphylaxis against FIX.
Adeno-associated Virus (AAV) Vector – Efficacy and Immunity
Adeno-associated virus (AAV) is a parvovirus with a single-stranded DNA genome of approximately 4.7 kb. It is a dependovirus that is unable to replicate in the absence of a helper virus such as adenovirus; thus, although it is a common natural infection, AAV is not associated with any known pathogenic infections in humans. Recombinant AAVs are modified by the removal of any DNA encoding for viral protein. Only the inverted terminal repeats (ITRs) required for packaging are retained from the viral genome, giving rAAV vectors a packaging capacity of about 5 kb for the promoter and gene of interest. Several factors make AAV an attractive vector for in vivo gene therapy, including its ability to infect nondividing cells, the maintenance of vector genomes as episomal concatemers (minimizing the risk of insertional mutagenesis), its relatively low immunogenicity and the wide variety of capsid serotypes that allow gene delivery to numerous target tissues and cell types.
Immune responses to AAV vectors have been observed in the clinic, posing a hurdle for gene therapy. The Herzog Lab continues to define the mechanisms of innate immunity to AAV vectors. For example, innate immunity in the liver was found to depend on sensing of the viral genome by the endosomal DNA receptor TLR9. Furthermore, TLR9-MyD88 pattern-recognition receptor pathway is uniquely capable of initiating CD8+ T cell responses against the viral capsid (while, for instance, TLR2, STING, or TLR4 have no effect). Importantly, both conventional (cDCs) and plasmacytoid dendritic cells (pDCs) are required for the cross-priming of capsid-specific CD8+ T cells, while other antigen presenting cells are not involved. TLR9 signaling is specifically required 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 cross-priming also require IFN I signaling and can be inhibited by administering specific molecules to prevent induction of capsid-specific CD8+ T cells. The effects of innate immune pathways immune responses to vector and transgene product in different target organs are under active investigation.
Treg and Oral Immunotherapy – Plants, Drugs, and Cells
A serious complication in replacement therapy for hemophilia is the formation of anti-drug antibodies (inhibitors) against the coagulation factor. Inhibitor formation against factor VIII (FVIII) in the treatment of hemophilia A occurs in 20-30 percent of patients. Although the incidence of inhibitor formation against factor IX (FIX, deficient in hemophilia B) is substantially lower, anaphylaxis and nephrotic syndrome complicate efforts to eradicate FIX inhibitors. No prophylactic clinical tolerance protocols exist for either clotting factor. To address this critical need, the Herzog and Daniell (Upenn) laboratories jointly developed an oral tolerance approach, which is based on transgenic crop plants expressing FVIII or FIX antigens in the chloroplasts of green leaves. The antigens are expressed as fusions to the transmucosal carrier CTB (cholera toxin B subunit), thus targeting a receptor on the gut epithelium for efficient delivery to the immune system following release in the small intestine. Prior to reaching the gut, the plant cell wall provides natural bioencapsulation that protects the antigens from degradation. Repeated oral delivery of lettuce prevented inhibitor formation and anaphylaxis against intravenous FIX in hemophilia B mice. Scale-up in a hydroponic system for production of clinical-grade material was successful. Recently, efficacy in a large, non-rodent animal model was demonstrated in studies in hemophilia B dogs. With regard to FVIII, oral tolerance induction was successful in hemophilia A mice using leaf cells expressing the heavy chain (HC) and C2 domain of human B domain-deleted (BDD) FVIII. Mechanistic studies show induction of two subsets of regulatory T cells (Treg) that suppress antibody formation, namely CD4+CD25+FoxP3+ Treg and LAP+CD4+CD25-FoxP3- Treg. The origin, interactions, and potential use as biomarkers of these Treg is under active investigation. In alternative approaches to tip the balance of the immune response to Treg induction, the Herzog lab developed short-term immune tolerance regimens based on co-administration of antigen, the mTOR inhibitor rapamycin, and synergistic drugs are being developed. Furthermore, tolerogenic cell therapies using gene modified B cells or ex vivo expanded Treg look promising.