Odelya E. Pagovich

AAVrh.10-mediated expression of an anti-cocaine antibody mediates persistent passive immunization that suppresses cocaine-induced behavior.

Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity.

Disrupted Adenovirus-Based Vaccines Against Small Addictive Molecules Circumvent Anti-Adenovirus Immunity

Adenovirus (Ad) vaccine vectors have been used for many applications due to the capacity of the Ad capsid proteins to evoke potent immune responses, but these vectors are often ineffective in the context of pre-existing anti-Ad immunity. Leveraging the knowledge that E1(-)E3(-) Ad gene transfer vectors are potent immunogens, we have developed a vaccine platform against small molecules by covalently coupling analogs of small molecules to the capsid proteins of disrupted Ad (dAd5). We hypothesized that the dAd5 platform would maintain immunopotency even in the context of anti-Ad neutralizing antibodies. To test this hypothesis, we coupled cocaine and nicotine analogs, GNE and AM1, to dAd5 capsid proteins to generate dAd5GNE and dAd5AM1, respectively. Mice were pre-immunized with Ad5Null, resulting in high titer anti-Ad5 neutralizing antibodies comparable to those observed in the human population. The dAd5GNE and dAd5AM1 vaccines elicited high anti-cocaine and anti-nicotine antibody titers, respectively, in both naive and Ad5-immune mice, and both functioned to prevent cocaine or nicotine from reaching the brain of anti-Ad immune mice. Thus, disrupted Ad5 evokes potent humoral immunity that is effective in the context of pre-existing neutralizing anti-Ad immunity, overcoming a major limitation for current Ad-based vaccines.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy

BACKGROUND Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. OBJECTIVE Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. METHODS We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. RESULTS A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. CONCLUSION If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.

Disease characteristics and progression in patients with late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease: an observational cohort study

BACKGROUND Late-infantile neuronal ceroid lipofuscinosis type 2 (CLN2) disease, characterised by rapid psychomotor decline and epilepsy, is caused by deficiency of the lysosomal enzyme tripeptidyl peptidase 1. We aimed to analyse the characteristics and rate of progression of CLN2 disease in an international cohort of patients. METHODS We did an observational cohort study using data from two independent, international datasets of patients with untreated genotypically confirmed CLN2 disease: the DEM-CHILD dataset (n=74) and the Weill Cornell Medical College (WCMC) dataset (n=66). Both datasets included quantitative rating assessments with disease-specific clinical domain scores, and disease course was measured longitudinally in 67 patients in the DEM-CHILD cohort. We analysed these data to determine age of disease onset and diagnosis, as well as disease progression-measured by the rate of decline in motor and language summary scores (on a scale of 0-6 points)-and time from first symptom to death. FINDINGS In the combined DEM-CHILD and WCMC dataset, median age was 35·0 months (IQR 24·0-38·5) at first clinical symptom, 37·0 months (IQR 35·0 -42·0) at first seizure, and 54·0 months (IQR 47·5-60·0) at diagnosis. Of 74 patients in the DEM-CHILD dataset, the most common first symptoms of disease were seizures (52 [70%]), language difficulty (42 [57%]), motor difficulty (30 [41%]), behavioural abnormality (12 [16%]), and dementia (seven [9%]). Among the 41 patients in the DEM-CHILD dataset for whom longitudinal assessments spanning the entire disease course were available, a rapid annual decline of 1·81 score units (95% CI 1·50-2·12) was seen in motor-language summary scores from normal (score of 6) to no function (score of 0), which occurred over approximately 30 months. Among 53 patients in the DEM-CHILD cohort with available data, the median time between onset of first disease symptom and death was 7·8 years (SE 0·9) years. INTERPRETATION In view of its natural history, late-infantile CLN2 disease should be considered in young children with delayed language acquisition and new onset of seizures. CLN2 disease has a largely predictable time course with regard to the loss of language and motor function, and these data might serve as historical controls for the assessment of current and future therapies. FUNDING EU Seventh Framework Program, German Ministry of Education and Research, EU Horizon2020 Program, National Institutes of Health, Nathans Battle Foundation, Cures Within Reach Foundation, Noahs Hope Foundation, Hope4Bridget Foundation.

755. One-time Gene Therapy for Hereditary Angioedema

Hereditary angioedema (HAE) is a potentially life-threatening autosomal dominant deficiency affecting 1 in 50,000 individuals from all ethnic groups worldwide. More than 99% of HAE cases are caused by a deficiency in functional plasma C1 esterase inhibitor (C1E-INH, a serine protease inhibitor) due to mutations in the SERPING1 gene. Low plasma C1E-INH activity dysregulates the contact, complement, and fibrinolytic systems, resulting in unpredictable, recurrent submucosal edema of cutaneous tissues, gastrointestinal and respiratory tracts. If not treated in a timely manner, laryngeal edema can result in death by asphyxiation. Current HAE treatments consist of management of acute attacks, repeated prophylactic therapy with C1E-INH or long term prophylaxis with attenuated androgens, each complicated by a high economic burden, limited compliance, drug side effects and contraindications. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of C1 esterase-inhibitor (serotype 10 expressing the human CIE-INH coding sequence, AAVrh.10hC1EI) would provide sustained C1E-INH activity levels in plasma, sufficient to prevent angioedema episodes. To study the efficacy of AAVrh.10hC1EI, using CRISPR/Cas9 technology we created a novel C1E-INH deficient mouse model analogous to human HAE disease, and characterized the resulting SERPING1 gene mutations by genome sequencing. The heterozygous mouse model shares characteristics associated with HAE in humans including decreased C1E-INH and C4 levels in plasma and increased vascular permeability. Administration of AAVrh.10hC1EI to the heterozygous mice resulted in sustained human C1E levels above the predicted therapeutic levels. In order to demonstrate that the increased vascular permeability observed in heterozygote C1E-INH+/- mice was a direct result of C1E-INH deficiency, Evans blue dye was injected intravenously and extravasation of dye from the vasculature evaluated. Compared to wild type mice under baseline conditions, the C1EINH+/- mice had increased extravasation of the dye into the hind paws quantitated by optical absorbance at 600 nm. Strikingly, AAVrh10.hC1EI-treated (1011 gc) mice displayed a marked decrease in dye extravasation, whereas non-treated C1E-INH +/- mice had markedly increased dye extravasation. These results demonstrate that a single treatment with AAVrh.10hC1EI has the potential to provide long term protection from angioedema attacks in the affected population, representing a paradigm shift in current therapeutic approaches.

Gene Therapy for C1 Esterase Inhibitor Deficiency in a Murine Model of Hereditary Angioedema

Hereditary angioedema (HAE) is a life‐threatening, autosomal dominant disorder characterized by unpredictable, episodic swelling of the face, upper airway, oropharynx, extremities, genitalia, and gastrointestinal tract. Almost all cases of HAE are caused by mutations in the SERPING1 gene resulting in a deficiency in functional plasma C1 esterase inhibitor (C1EI), a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems. Current treatment of HAE includes long‐term prophylaxis with attenuated androgens or human plasma‐derived C1EI and management of acute attacks with human plasma‐derived or recombinant C1EI, bradykinin, and kallikrein inhibitors, each of which requires repeated administration. As an approach to effectively treat HAE with a single treatment, we hypothesized that a one‐time intravenous administration of an adeno‐associated virus (AAV) gene transfer vector expressing the genetic sequence of the normal human C1 esterase inhibitor (AAVrh.10hC1EI) would provide sustained circulating C1EI levels sufficient to prevent angioedema episodes.

36. Translation of an Adenovirus-Based Cocaine Vaccine dAd5GNE to a Clinical Trial

dAd5GNE, an anti-cocaine vaccine based on a disrupted serotype 5 adenovirus gene transfer vector covalently conjugated to the cocaine analog GNE, evokes high titers of high affinity anti-cocaine antibodies that prevent cocaine from reaching its cognate receptor in the CNS. dAd5GNE has been shown to be effective in preclinical efficacy studies in mice, rats and nonhuman primates. In order to translate dAd5GNE to the clinic, this study focused on optimizing the choice of adjuvant, timing of vaccination regimen and dose. Adjuvant. To evaluate vaccine formulation, the adjuvants Adjuplex and Alum were assessed at a fixed dose to identify the formulation that evoked the fastest and highest titer response. BALB/c mice vaccinated with dAd5GNE/Adjuplex demonstrated high serum anti-cocaine antibody titers (>5×105) after a single administration while animals vaccinated with dAd5GNE/Alum required multiple injections to achieve high titers, and mice vaccinated with dAd5GNE/PBS plateaued at a lower titer. Therefore, a formulation based on the Adjuplex adjuvant was chosen for the clinical protocol. Timing. The measurement of titer half-life in nonhuman primates following individual vaccinations at variable intervals was used to inform the timing between vaccine boosts. The anti-cocaine antibody titer half-life in 8 dAd5GNE/Adjuplex vaccinated nonhuman primates was assayed regularly over the course of 1 yr and the titer half-life was calculated following each administration. The average half-life for all animals was 4.0 ± 0.2 wk. Continued cocaine use did not impact the titer half-life. These results indicate that a 4 wk interval for vaccinations is necessary to maintain the high titer anti-cocaine antibody levels. Dose. We measured the dose response in mice to provide the baseline for a phase I/II clinical trial and identified the range of doses that bracket the anticipated minimum effective dose and maximum tolerated dose. Doses from 0.04-40 μg of the vaccine were evaluated in BALB/c for the capacity to evoke antibody titers and to minimize access of radiolabelled cocaine to the CNS. Doses at and above 4 µg produced high anti-cocaine antibody titers above the threshold for efficacy (>5×105) which substantially reduced cocaine levels in the brain (p<0.0001 vs PBS). Using a modified weight adjusted dose from these results, the vaccine doses to be evaluated in the clinical trial were determined to be 100 to 1000 µg. The results of these studies combine to provide the specifications for critical vaccine design parameters required to translate the anti-cocaine vaccine to evaluation in a clinical trial. Based on these specifications the FDA has allowed the Adjuplex-formulated dAd5GNE vaccine to proceed to clinical study with a monthly vaccination regimen in recovering cocaine addicts.

471. In Vivo Potency Assay for AAV-Based Gene Therapy Vectors

One hurdle in the translation of adeno-associated virus (AAV) vectors for the treatment of human disease is the challenge in demonstrating drug function in a potency assay, a requirement in the clinical translation to product. In vitro measurements of AAV potency suffer from poor efficiency of infectivity and are insensitive measures of potency. To address this issue, we have developed a robust in vivo assay for the measurement of vector potency. Using as the model vector the AAVrh.10 serotype coding for human frataxin (FXN, a mitochondrial protein essential for cellular function and that is deficient in Friedreichs ataxia), and based on the knowledge that intravenous administration of all AAV vectors primarily transduce the liver, we have developed a reproducible in vivo potency assay that can be used to set quality control standards for vector production. The assay is based on administration of the AAV vector (2.5 × 1010 genome copies) administered intravenously to 6 to 8 wk old Balb/c male mice with the liver harvested 2 wk later following PBS perfusion. Liver homogenates are processed to assess vector genome copies, transgene mRNA, and expressed protein. In order to establish acceptance criteria for assays of vector genome and mRNA levels, separate specifications were set for DNA sample load using the mouse housekeeping gene Tfrc, and RNA sample load using mouse 18S RNA, both based on data from quantitative PCR analysis of mouse livers (n=35) assayed in duplicate. Specifications were set as the median ± 2 standard deviations based on these assay results; for the potency assay results to be accepted, each of these specifications must be met by the test sample. For the AAVrh. 10 vector expressing FXN(AAVrh.10hFXN), liver vector genome levels (a measure of reproducibility of delivery) were 8.2 × 104 ± 2.2 × 104 genome copies/µg genomic DNA (n=10 mice, mean ± SD), liver human FXN mRNA levels (a measure of vector potency at the transcription level) were 2.2 × 103 ± 0.7 × 103 copies/µg total RNA (n=10 mice, mean ± SD) and liver human FXN protein levels (ELISA; a measure of vector potency at the protein level) were 54.8 ± 17.52 ng/mg protein (n=10 mice, mean ± SD). From this data, we established the following quality control specifications for AAVrh.10hFXN vectors: vector genome range 6 × 104 - 1 × 105 (copies/µg DNA), hFXN mRNA level range 1.6 × 103 - 2.9 × 103 (copies/µg RNA) and the hFXN protein levels 37 - 72 (ng/mg protein). This approach is adaptable to any AAV vector, providing an in vivo quality control for vector function.

444. One-Time Gene Therapy to Prevent Peanut-Induced Anaphylaxis

Peanut allergy is a major public health problem that accounts for the majority of fatal food-induced anaphylactic reactions. There are no curative or preventative therapies. Therapy with omalizumab, a human anti-IgE monoclonal antibody, is transiently effective, but it must be administered parenterally, has a short half-life and is costly, thus it is challenging to use as a preventative for peanut allergy. To circumvent this challenge, we hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of omalizumab would provide persistent high circulating levels of anti-IgE sufficient to protect against repeated peanut exposure in the peanut allergic host. To assess this hypothesis, we created a humanized murine model of peanut allergy by reconstituting NOD-scid IL2rgnull mice with 3 x107 blood mononuclear cells (PBMC) derived from peanut allergic individuals or non-allergic controls and treating the mice with a non-human primate serotype rh.10 AAV expressing omalizumab (AAVrh.10anti-IgE) or control vector. Administration of a single dose (1×1011 gc) of AAVrh.10anti-IgE resulted in persistent high levels of anti-IgE in both wild type and humanized mouse models, with anti-IgE levels > 400 mg/ml at 6 wk and remained at similar levels to the last time point evaluated at 24 wk in wild type mice and levels above 220 mg/ml were achieved in NSG mice at wk 8. No anti-IgE was detected in serum from mice that received a non-relevant control vector or PBS alone. The humanized NOD-scid IL2rgnull mice were sensitized intraperitoneally with crude peanut extract (CPE) weekly for 4 wk and then challenged with CPE via oral gavage. Total serum human IgG, IgE and peanut-specific IgE were measured weekly. After challenge, allergic phenotype was assessed using an anaphylaxis score, histamine levels, rectal temperature and behavior. All NSG mice were successfully reconstituted with mononuclear cells as measured by persistent levels of human IgG. Mice treated with AAVrh.10anti-IgE had elevated total and peanut-specific IgE from day 28 onwards with total IgE lower in the AAVrh.10anti-IgE treated mice. Strikingly, after peanut challenge, AAVrh.10anti-IgE treated mice displayed a less severe allergic phenotype compared to those that received the control vector as defined by decreased histamine release, a lower anaphylaxis score, no drop in rectal temperature and suppressed ambulation. These results demonstrate that a single treatment with AAVrh.10anti-IgE provides long term protection from peanut allergen challenge in the peanut sensitized population, representing a paradigm shift in current therapeutic approaches.