Download Oral Delivery of the Factor VIII Gene: Immunotherapy for Hemophilia A

2011 Research Grant Program Winning Abstract
Oral Delivery of the Factor VIII Gene: Immunotherapy for Hemophilia A
By Chris Grigsby
The X-linked bleeding disorder hemophilia A (coagulation factor VIII, FVIII, deficiency)
affects 1 in 5,000 males worldwide. Following treatment with recombinant or plasmaderived FVIII, 20 to 30% of hemophilia A patients form inhibitory antibodies (inhibitors), a
serious complication that increases morbidity and mortality. Immune tolerance induction
protocols consist of high-dose factor administrations for a long period of time (months to
years), are very expensive (>$1,000,000), and often have to be stopped due to
anaphylactic reactions or nephritic complications.
Protein antigen delivery to the mucosal immune system in the gut is tolerogenic and
restricts inhibitor formation without immune suppression and without a need for T-cell
epitope mapping. Alternatively, the gene for the antigen can be delivered to the intestinal
epithelium for oral immunization, where its sustained expression results in continuous
exposure to tolerogenic antigen. Microfold (M) cells in the epithelial layer facilitate
antigen presentation and B- and T- cell activation by transporting antigen to the
subepithelial lymphoid tissues that harbor lymphocytes, dendritic cells (DCs), and
macrophages. We have employed chitosan, a natural mucoadhesive polysaccharide, to
deliver FVIII DNA packaged into nanoparticles for both immune modulation and
phenotypic correction of the disease.
Preliminary Data
In a murine knockout model, feeding of chitosan-FVIII DNA nanoparticles in gelatin
mediated a prolonged restoration of hemostasis. Functional FVIII protein was detected in
plasma, reaching a peak level of 2–4% normal FVIII at day 22. A bleeding challenge
after one month resulted in survival of 13 of 20 mice compared to 0 of 6 untreated mice.
In a recent pilot study, the same particles were applied to an inhibitor-positive hemophilic
dog. While dosing did not restore hemostasis, the inhibitors were eradicated. The total
ablation of inhibitors remained durable beyond five months, even following FVIII
administration to treat hemorrhages. We have also previously immunized mice using
orally administered chitosan nanoparticles carrying the dust mite allergen gene (Derp1)
and the peanut allergen gene (Arah2). The studies proposed next aim to elucidate the
immunological and delivery mechanisms underlying these phenomena to improve both
tolerogenesis and therapeutic potential.
Aim 1
To maximize targeted delivery and gene transfer, we will coat the chitosan nanoparticles
with Eudragit®, a pH-sensitive polymer that is insoluble in the acidic environment of the
stomach but dissolves under the high pH in the small intestines. Chitosan is nontoxic
(LD50 over 16 g/kg in mice), but the toxicity of each macroformulation will be measured
by flow cytometry using PI and Annexin V apoptosis assays. Confirmation of uptake of
quantum dot labeled nanoparticles in Peyer’s patches containing M cells and CD11c+
DCs will be accomplished in a rat closed-loop intestinal model using
immunohistochemistry and flow cytometry. We will quantify paracellular transepithelial
transport by co-localization of nanoparticles with tight junction proteins ZO-1 and
occludin versus transcytosis with endosomal markers such as EEA1 and Rab5. Uptake
and transfection of enterocytes, M cells, and immune cells will be differentially detected
using a GFP reporter gene and multicolor flow cytometry.
Aim 2
Optimized nanoparticles packaged in Eudragit-coated gelatin capsules will be
administered in a canine hemophilia model to study therapeutic efficacy and immune
modulation in parallel. Feeding of antigen can activate CD4+ T cells, generating an
immune regulatory and anti-inflammatory response. Intestinal regulatory T cells such as
CD4+CD25+FoxP3+ cells, Th3, or Tr1 cells secrete immune-suppressive cytokines (IL-10
and TGF-B), which can inhibit DC and T-cell activation. Certain subsets of antigenpresenting cells (APCs) such as CD103+ DCs, plasmacytoid DCs (pDCs), and
tolerogenic B cells, participate in promoting immune regulation in the gut and will be
studied. We will use whole blood clotting times, activated partial thromboplastin times,
FVIII activity (chromogenic assay/coatest), and inhibitor titers (Bethesda assay, Ig
subclass-specific ELISA) to monitor phenotypic correction and immune responses.
Further analyses of plasma samples will quantify titers of Th2-dependent IgG1 and IgE,
TGF-B– dependent IgA and IgG2b, and secretory IgA in fecal extracts indicative of a
mucosal immune response.
In vitro re-stimulation of splenocyte cultures with FVIII will determine if the T-cell
response is dominated by IL-4 production, the Th2 cytokine that drives class switching to
IgG1 and IgE, and if IL-10 or TGF-B is present. We hypothesize that our oral
nanoparticle immunotherapy will suppress the IL-4 response and instead induce FoxP3,
IL-10, and TGF-B expression. To assess targeting and transfection of the gut-associated
lymphoid tissue (GALT) constituents, intestine will be harvested for
immunohistochemistry and flow cytometry. Antibody and other stains will include those
for FVIII, DCs (CD11c, CD103), macrophages (F4/80), M cells (UEA-1), and B cells
(CD19). Functionally, immunized and naïve animals will be challenged for onset and
severity of anaphylactic response to FVIII. Nanoparticle therapy is expected to prevent
anaphylaxis, IgE formation, and inhibitor formation, and to suppress IL-4 production
while promoting IL-10, TGF-B, and FoxP3 expression.
Significance
Having achieved phenotypic correction in mice and inhibitor ablation in a pilot canine
study, the goal of an entirely oral-based hemophilia A treatment regimen that
accomplishes prophylaxis against bleeds and prevents inhibitor formation is tangible and
could readily be adapted to other inherited protein deficiencies. Financial support from
BD would greatly enable this translational proposal.
The BD Biosciences Research Grant Program aims to reward and enable important
research by providing vital funding for scientists pursuing innovative experiments to
advance the scientific understanding of disease.
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