Abstract: Gene transfer for hemophilia A offers the potential for a one-time disease altering treatment, eliminating the risk of bleeds while freeing patients from the burden of lifelong chronic therapy. SPK-8011 consists of a bioengineered AAV capsid expressing B domain-deleted factor VIII (FVIII) under the control of a liver-specific promoter. In pre-clinical studies, we showed a dose-dependent increase in circulating FVIII levels in non-human primates infused with SPK-8011. We conducted a Phase I/II study of SPK-8011 in 12 men (ages 18-52 years) with severe (n=11) or moderately severe (n=1) hemophilia A. Prior to gene therapy, 8/12 subjects were on prophylaxis, and 4/12 received on-demand treatment. Subjects were enrolled in 1 of 3 dose cohorts, 5E11 vg/kg (n=2), 1E12(n=3), or 2E12(N=7). Safety analysis showed no inhibitor formation. A single serious adverse event (SAE) was reported, associated with an immune response to AAV capsid characterized by simultaneous decline in FVIII, transaminase elevation peaking at Grade 2, and development of positive IFN-g ELISPOTs to capsid was observed beginning at week 6.5 after vector infusion. The asymptomatic transaminase elevation did not respond promptly to initiation of oral steroids and the subject received two infusions of IV methylprednisolone in hospital, thereby fulfilling SAE criteria. The SAE has resolved. All vector doses led to expression of FVIII levels adequate to prevent bleeding and allow cessation of prophylaxis. Across the 12 subjects at 3 doses, there was a 97% reduction in annualized bleeding rate (ABR), and a 97% reduction in annualized infusion rate (AIR). In the 5E11 dose cohort, mean FVIII levels beginning 12 weeks post vector infusion are 13%, with no bleeding events, no elevated transaminase levels, no use of steroids, and stable FVIII expression out to 66 weeks (ongoing). In the 1E12 dose cohort, mean FVIII levels are 15% beginning at 12 weeks post-infusion and stable out to 46 weeks (ongoing). The first subject in the 1E12 dose infused a single dose of factor concentrate for a spontaneous joint bleed at day 159, and the second received multiple infusions for a traumatic bleed beginning at day 195. Declining FVIII levels triggered initiation of a course of tapering steroids in both subjects, at 12 and 7 weeks post vector infusion respectively, which led to stabilization of FVIII levels. The third subject has had no bleeding and did not receive factor infusions or steroids. In the 2E12 (highest) dose cohort, 5/7 subjects currently have FVIII levels 16-49%; their mean FVIII level beginning 12 weeks post-infusion is 30%. No bleeds have been reported among these subjects beginning 4 weeks post vector infusion. Additionally, 5/7 subjects in the 2E12 dose cohort received a course of steroids, initiated at 6-11 weeks post vector infusion, for one or more of the following: declining FVIII levels, rise in ALT above subject baseline, or elevated IFN-g ELISPOTs to AAV capsid. Steroid initiation normalized ALT levels and extinguished the ELISPOT signal in all cases; 2 subjects showed limited stabilization of FVIII levels, which fell to 〈 6% likely due to the immune response. For one of these, no bleeds have been reported through 12 weeks of follow up; the other has had 4 bleeds through 37 weeks of observation. Our data indicate that the kinetics of SPK-8011 expression are similar to those observed with investigational SPK-9001 for hemophilia B. All subjects demonstrated durable transgene expression for up to 66 weeks post vector administration (data cutoff 7/13/18). On cumulative follow up of 345 weeks, SPK-8011 demonstrated a favorable safety profile with no evidence of FVIII inhibitor formation, a single SAE, and 2/12 subjects who experienced ALT elevation above the upper limit of normal that resolved with steroid initiation. Data from the 5E11 (lowest) dose cohort are consistent with published natural history data indicating FVIII:C 12% is adequate to prevent spontaneous bleeding events. Given that 2 subjects in the 2E12 dose cohort lost some FVIII expression, which then stabilized on steroids, and 5/7 subjects in this cohort required steroids, prophylactic steroids may be warranted. We conclude that infusion of SPK-8011 in 12 subjects with severe or moderately severe hemophilia A resulted in safe, durable, dose-dependent FVIII expression resulting in an excellent preliminary efficacy profile with an overall 97% reduction in ABR and AIR. Disclosures High: Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. George:University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. Ragni:CSL Behring: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sangamo: Research Funding; Shire: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; SPARK: Consultancy, Research Funding. Croteau:Novo Nordisk: Consultancy; Octapharma: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Spark Therapeutics: Research Funding; Tremeau Pharmaceuticals: Consultancy; Genetech: Consultancy, Research Funding; CSL-Behring: Consultancy; Catalyst Biosciences: Consultancy; Bioveritiv: Consultancy; Biomarin: Consultancy; Bayer: Consultancy; Baxalta/Shire: Consultancy, Research Funding. Joseney-Antoine:Spark Therapeutics: Employment. Macdougall:Spark Therapeutics: Employment. Tompkins:Spark Therapeutics: Employment. Hait:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Bassiri:Spark Therapeutics: Employment. Valentino:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Mingozzi:Spark Therapeutics, Inc.: Employment. Anguela:Spark Therapeutics, Inc.: Employment. Reape:Spark Therapeutics: Employment.

Abstract: Recent studies showed that enteric helminth infection improved symptoms in patients with inflammatory bowel disease as well as in experimental models of colitis. The aim of this study was to determine the mechanism of the protective effect of helminth infection on colitis-induced changes in immune and epithelial cell function. BALB/c mice received an oral infection of Heligmosomoides polygyrus third-stage larvae, were given intrarectal saline or trinitrobenzene sulfonic acid (TNBS) on day 10 postinfection, and were studied 4 days later. Separate groups of mice received intrarectal saline or TNBS on day 10 and were studied on day 14. Muscle-free colonic mucosae were mounted in Ussing chambers to measure mucosal permeability and secretion. Expression of cytokines was assessed by quantitative real-time PCR, and mast cells were visualized by immunohistochemistry. TNBS-induced colitis induced mucosal damage, upregulated Th1 cytokines, and depressed secretory responses. Heligmosomoides polygyrus elevated Th2 cytokine expression, increased mast cell infiltration and mucosal resistance, and also reduced some secretory responses. Prior H. polygyrus infection prevented TNBS-induced upregulation of Th1 cytokines and normalized secretory responses to specific agonists. TNBS-induced colitis did not alter H. polygyrus -induced mast cell infiltration or upregulation of Th2 cytokine expression. The results indicate that the protective mechanism of enteric nematode infection against TNBS-induced colitis involves prevention of Th1 cytokine expression and improved colonic function by a mechanism that may involve mast cell-mediated protection of neural control of secretory function. Similar response patterns could account for the clinical improvement seen in inflammatory bowel disease with helminthic therapy.

Abstract: Gastrointestinal nematode infections generally invoke a type 2 cytokine response, characterized by the production of IL-4, IL-5, IL-9, and IL-13. Among these cytokines, IL-4 and IL-13 exhibit a functional overlap that can be explained by the sharing of a common receptor or receptor component (IL-4Rα). Binding of IL-4 by either the type 1 or 2 IL-4R, or of IL-13 by the type 2 IL-4R, initiates Jak-dependent tyrosine phosphorylation of the IL-4Rα-chain and the transcription factor, STAT6. In the present study, we investigated: 1) whether IL-13 has effects on intestinal epithelial cells similar to those observed with IL-4, and 2) whether the effects of IL-4 and IL-13 depend on STAT6 signaling and/or mast cells. BALB/c, STAT6−/−, and mast cell-deficient W/Wv mice or their +/+ littermates were treated with a long-lasting formulation of recombinant mouse IL-4 (IL-4C) or with IL-13 for seven days. Segments of jejunum were mounted in Ussing chambers to measure mucosal permeability; chloride secretion in response to PGE2, histamine, 5-hydroxytryptamine, or acetylcholine; and Na+-linked glucose absorption. IL-4C and IL-13 increased mucosal permeability, decreased glucose absorption, and decreased chloride secretion in response to 5-hydroxytryptamine. These effects were dependent on STAT6 signaling. Responses to PGE2 and histamine, which were dependent on mast cells and STAT6, were enhanced by IL-4C, but not by IL-13. The effects of IL-4 and IL-13 on intestinal epithelial cell function may play a critical role in host protection against gastrointestinal nematodes.

Abstract: ▪ Abstract  Studies with rodents infected with Trichinella spiralis, Heligmosomoides polygyrus, Nippostrongylus brasiliensis, and Trichuris muris have provided considerable information about immune mechanisms that protect against parasitic gastrointestinal nematodes. Four generalizations can be made: 1. CD4 + T cells are critical for host protection; 2. IL-12 and IFN-γ inhibit protective immunity; 3. IL-4 can: (a) be required for host protection, (b) limit severity of infection, or (c) induce redundant protective mechanisms; and 4. Some cytokines that are stereotypically produced in response to gastrointestinal nematode infections fail to enhance host protection against some of the parasites that elicit their production. Host protection is redundant at two levels: 1. IL-4 has multiple effects on the immune system and on gut physiology (discussed in this review), more than one of which may protect against a particular parasite; and 2. IL-4 is often only one of multiple stimuli that can induce protection. Hosts may have evolved the ability to recognize features that characterize parasitic gastrointestinal nematodes as a class as triggers for a stereotypic cytokine response, but not the ability to distinguish features of individual parasites as stimuli for more specific protective cytokine responses. As a result, hosts deploy a set of defense mechanisms against these parasites that together control infection by most members of that class, even though a specific defense mechanism may not be required to defend against a particular parasite and may even damage a host infected with that parasite.

Abstract: IL-4 and IL-13 promote gastrointestinal worm expulsion, at least in part, through effects on nonlymphoid cells, such as intestinal epithelial cells. The role of IL-4/IL-13 in the regulation of intestinal epithelial function during Heligmosomoides polygyrus (Hp) infection was investigated in BALB/c mice infected with Hp or treated with a long-lasting formulation of recombinant mouse IL-4/αIL-4 complexes (IL-4C) for 7 days. Separate groups of BALB/c mice were drug-cured of initial infection and later reinfected and treated with anti-IL-4R mAb, an antagonist of IL-4 and IL-13 receptor binding, or with a control mAb. Segments of jejunum were mounted in Ussing chambers, and short circuit current responses to acetylcholine, histamine, serotonin, PGE2, and glucose were determined. Although only modest changes in epithelial cell function were observed during primary Hp infection, IL-4C or a secondary Hp infection each induced more dramatic changes, including increased mucosal permeability, reduced sodium-linked glucose absorption, and increased Cl− secretory response to PGE2. Some, but not all, effects of IL-4C and Hp infection were dependent on enteric nerves. Hp-induced changes in epithelial function were attenuated or prevented by anti-IL-4R mAb. Thus, IL-4/IL-13 mediate many of the effects of Hp infection on intestinal epithelial cell function and do so both through direct effects on epithelial cells and through indirect, enteric nerve-mediated prosecretory effects. These immune system-independent effector functions of IL-4/IL-13 may be important for host protection against gastrointestinal nematodes.