Gene Therapy for Immunoglobulin E, Complement-Mediated, and Eosinophilic Disorders.

Immunoglobulin E, complement, and eosinophils play an important role in host defense, but dysfunction of each of these components can lead to a variety of human disorders. In this review, we summarize how investigators have adapted gene therapy and antisense technology to modulate immunoglobulin E, complement, and/or eosinophil levels to treat these disorders.

Gene Therapy to Treat Osteopenia Associated With Chronic Ethanol Consumption and Aldehyde Dehydrogenase 2 Deficiency.

Aldehyde dehydrogenase 2 (ALDH2) deficiency affects 35% to 45% of East Asians and 8% of the world population. ALDH2 is the second enzyme in the ethanol metabolism pathway. The common genetic variant ALDH2*2 allele has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the enzyme activity, resulting in an accumulation of acetaldehyde after ethanol consumption. The ALDH2*2 allele is associated with increased risk of osteoporosis and hip fracture. Our prior study showed that administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) before initiation of ethanol consumption prevented bone loss in ALDH2-deficient homozygous knockin mice carrying the E487K mutation (Aldh2 E487K+/+). We hypothesized that AAVrh.10hALDH2 administration after establishment of osteopenia would be able to reverse bone loss due to ALDH2 deficiency and chronic ethanol consumption. To test this hypothesis, male and female Aldh2 E487K+/+ mice (n = 6) were given ethanol in the drinking water for 6 weeks to establish osteopenia and then administered AAVrh.10hALDH2 (1011 genome copies). Mice were evaluated for an additional 12 weeks. AAVrh.10hALDH2 administration after osteopenia was established corrected weight loss and locomotion phenotypes and, importantly, increased midshaft femur cortical bone thickness, the most important component of bone in the resistance to fractures, and showed a trend toward increased trabecular bone volume. AAVrh.10hALDH2 is a promising therapeutic for osteoporosis in ALDH2-deficient individuals. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Gene therapy in a murine model of chronic eosinophilic leukemia-not otherwise specified (CEL-NOS).

Chronic eosinophilic leukemia-not otherwise specified (CEL-NOS) is a rare, aggressive, fatal disease characterized by blood eosinophilia and dysfunction of organs infiltrated with eosinophils. Clinically, the disease manifests with weight loss, cough, weakness, diarrhea, and multi-organ dysfunction that is unresponsive to therapy. We developed a one-time gene therapy for CEL-NOS using an adeno-associated virus (AAV) expressing an anti-eosinophil monoclonal antibody (AAVrh.10mAnti-Eos) to provide sustained suppression of eosinophil numbers in blood, thus reducing eosinophil tissue invasion and organ dysfunction. A novel CEL-NOS model was developed in NOD-scid IL2rγnull (NSG) mice by administration of AAV expressing the cytokine IL5 (AAVrh.10mIL5), resulting in marked peripheral and tissue eosinophilia of the heart, lung, liver, and spleen, and eventually death. Mice were administered AAVrh.10mAnti-Eos (1011 genome copies) 4 wk after administration of AAVrh.10mIL5 and evaluated for anti-eosinophil antibody expression, blood eosinophil counts, organ eosinophil invasion, and survival. AAVrh.10mAnti-Eos expressed persistent levels of the anti-eosinophil antibody for >24 wk. Strikingly, CEL-NOS treated mice had markedly lower blood eosinophil levels and reduced mortality when compared with control treated mice. These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide substantial therapeutic benefit to patients with CEL-NOS, a fatal malignant disorder.

Gene therapy for a murine model of eosinophilic esophagitis.

BACKGROUND: Eosinophils are specialized granulocytic effector cells that store and release highly active mediators used in immune defense. Eosinophils are also implicated in the pathogenesis of allergic disorders, including eosinophilic esophagitis (EoE), a chronic disorder characterized by infiltration of eosinophils into the esophagus and release of mediators that damage tissue, resulting in gastrointestinal morbidity, food impaction, and dysphagia. Treatment with elimination diets and/or topical corticosteroid therapy slow disease progression, but are complicated by adverse effects, limited compliance, and loss of response to therapy. We hypothesized that a single administration of an adeno-associated virus (AAV) coding for an anti-eosinophil monoclonal antibody that induces eosinophil clearance (anti-Siglec-F) would treat on a persistent basis a murine model of EoE. METHODS: A mouse model of peanut-induced EoE that mimics the human disease was established by sensitization and challenge with peanut extract. After challenge, these mice exhibited an EoE phenotype demonstrated by elevated levels of blood eosinophils, infiltration of eosinophils in the esophagus with associated esophageal remodeling and food impaction. RESULTS: The mice were treated with a single intravenous administration (1011 genome copies) of AAVrh.10mAnti-Eos, a serotype rh.10 AAV vector coding for an anti-Siglec-F monoclonal antibody. Vector administration resulted in persistent, high levels of anti-Siglec-F antibody expression. Administration of AAVrh.10mAnti-Eos to the mouse model of EoE reduced blood (P < 0.02) and esophageal eosinophil numbers (P < 0.002) protected from esophageal tissue remodeling and minimized food impaction. CONCLUSION: These results suggest that a single treatment with AAVrh.10mAnti-Eos has the potential to provide persistent therapeutic benefit to patients with EoE.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Systemic Adeno-Associated Virus-Mediated Gene Therapy Prevents the Multiorgan Disorders Associated with Aldehyde Dehydrogenase 2 Deficiency and Chronic Ethanol Ingestion.

Aldehyde dehydrogenase type 2 (ALDH2), a key enzyme in ethanol metabolism, processes toxic acetaldehyde to nontoxic acetate. ALDH2 deficiency affects 8% of the world population and 35-45% of East Asians. The ALDH2*2 allele common genetic variant has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the oxidizing ability of the enzyme resulting in systemic accumulation of acetaldehyde with ethanol ingestion. With chronic ethanol ingestion, mutations in ALDH2 are associated with a variety of hematological, neurological, and dermatological abnormalities, and an increased risk for esophageal cancer and osteoporosis. Based on our prior studies demonstrating that a one-time administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) prevents the acute effects of ethanol administration (the "Asian flush syndrome"), we hypothesized that AAVrh.10hALDH2 would also prevent the chronic disorders associated with ALDH2 deficiency and chronic ethanol ingestion. To assess this hypothesis, AAVrh.10hALDH2 (1011 genome copies) was administered intravenously to two models of ALDH2 deficiency, Aldh2 knockout homozygous (Aldh2-/-) and knockin homozygous (Aldh2E487K+/+) mice (n = 10 per group). Four weeks after vector administration, mice were given drinking water with 10-15% ethanol for 12 weeks. Strikingly, compared with nonethanol drinking littermates, AAVrh.10hALDH2 administration prevented chronic ethanol-induced serum acetaldehyde accumulation and elevated liver malondialdehyde levels, loss of body weight, reduced hemoglobin levels, reduced performance in locomotor activity tests, accumulation of esophageal DNA damage and DNA adducts, and development of osteopenia. AAVrh.10hALDH2 should be considered as a preventative therapy for the increased risk of chronic disorders associated with ALDH2 deficiency and chronic alcohol exposure.

Gene Therapy Correction of Aldehyde Dehydrogenase 2 Deficiency.

Aldehyde dehydrogenase 2 (ALDH2) deficiency causes "Asian flush syndrome," presenting as alcohol-induced facial flushing, tachycardia, nausea, and headaches. One of the most common hereditary enzyme deficiencies, it affects 35%-40% of East Asians and 8% of the world population. ALDH2 is the key enzyme in ethanol metabolism; with ethanol challenge, the common ALDH2*2 (E487K) mutation results in accumulation of toxic acetaldehyde. ALDH2*2 heterozygotes have increased risk for upper digestive tract cancers, compounded by smoking and drinking alcohol. We hypothesized that a one-time administration of an adeno-associated virus (AAV) gene transfer vector expressing the human ALDH2 coding sequence (AAVrh.10hALDH2) would correct the deficiency state. AAVrh.10hALDH2 was administered intravenously to Aldh2 knockout (Aldh2 -/-) and Aldh2 E487K knockin homozygous (Aldh2 E487K+/+) mice. Following acute ethanol ingestion, untreated ALDH2-deficient mice had elevated acetaldehyde levels and performed poorly in behavioral tests. In contrast, treated Aldh2 -/- and Aldh2 E487K+/+ mice had lower serum acetaldehyde levels and improved behavior. Thus, in vivo AAV-mediated ALDH2 therapy may reverse the deficiency state in ALDH2*2 individuals, eliminating the Asian flush syndrome and reducing the risk for associated disorders.

Pathogenic Autoreactive T and B Cells Cross-React with Mimotopes Expressed by a Common Human Gut Commensal to Trigger Autoimmunity.

Given the immense antigenic load present in the microbiome, we hypothesized that microbiota mimotopes can be a persistent trigger in human autoimmunity via cross-reactivity. Using antiphospholipid syndrome (APS) as a model, we demonstrate cross-reactivity between non-orthologous mimotopes expressed by a common human gut commensal, Roseburia intestinalis (R. int), and T and B cell autoepitopes in the APS autoantigen ß2-glycoprotein I (ß2GPI). Autoantigen-reactive CD4+ memory T cell clones and an APS-derived, pathogenic monoclonal antibody cross-reacted with R. int mimotopes. Core-sequence-dependent anti-R. int mimotope IgG titers were significantly elevated in APS patients and correlated with anti-ß2GPI IgG autoantibodies. R. int immunization of mice induced ß2GPI-specific lymphocytes and autoantibodies. Oral gavage of susceptible mice with R. int induced anti-human ß2GPI autoantibodies and autoimmune pathologies. Together, these data support a role for non-orthologous commensal-host cross-reactivity in the development and persistence of autoimmunity in APS, which may apply more broadly to human autoimmune disease.

Gene therapy for C1 esterase inhibitor deficiency in a Murine Model of Hereditary angioedema.

BACKGROUND: 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. METHODS: To study the efficacy of AAVrh.10hC1EI, we used CRISPR/Cas9 technology to create a heterozygote C1EI-deficient mouse model (S63±) that shares characteristics associated with HAE in humans including decreased plasma C1EI and C4 levels. Phenotypically, these mice have increased vascular permeability of skin and internal organs. RESULTS: Systemic administration of AAVrh.10hC1EI to the S63± mice resulted in sustained human C1EI activity levels above the predicted therapeutic levels and correction of the vascular leak in skin and internal organs. CONCLUSION: A single treatment with AAVrh.10hC1EI has the potential to provide long-term protection from angioedema attacks in affected individuals.

Corneal confocal microscopy: Neurologic disease biomarker in Friedreich ataxia.

OBJECTIVE: Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by mutations in the gene encoding for the mitochondrial protein frataxin, is characterized by ataxia and gait instability, immobility, and eventual death. We evaluated corneal confocal microscopy (CCM) quantification of corneal nerve morphology as a novel, noninvasive, in vivo quantitative imaging biomarker for the severity of neurological manifestations in FRDA. METHODS: Corneal nerve fiber density, branch density, and fiber length were quantified in individuals with FRDA (n = 23) and healthy age-matched controls (n = 14). All individuals underwent genetic testing and a detailed neurological assessment with the Scale for the Assessment and Rating of Ataxia (SARA) and Friedreich's Ataxia Rating Scale (FARS). A subset of individuals with FRDA who were ambulatory underwent quantitative gait assessment. RESULTS: CCM demonstrated a significant reduction in nerve fiber density and length in FRDA compared to healthy controls. Importantly, CCM parameters correlated with genotype, SARA and FARS neurological scales, and linear regression modeling of CCM nerve parameter-generated equations that predict the neurologic severity of FRDA. INTERPRETATION: Together, the data suggest that CCM quantification of corneal nerve morphology is a rapid, sensitive imaging biomarker for quantifying the severity of neurologic disease in individuals with FRDA. Ann Neurol 2018;84:893-904.

THE JEREMIAH METZGER LECTURE NOVEL THERAPEUTIC STRATEGIES OF ALLERGIC AND IMMUNOLOGIC DISORDERS.

Advances in understanding the immunological basis and mechanisms underlying allergic and immunologic disorders have led to effective but costly long-term and repetitive biologic therapies. Gene therapy is a rapidly advancing technology, in which a single administration of an adeno-associated virus encoding the therapeutic protein or monoclonal antibody may provide effective long-term therapy for allergic and immunologic disorders. In this review, we summarize the recent studies from our laboratory developing gene therapy strategies to treat hereditary angioedema and peanut allergy. The unraveling of the pathogenesis of immune-based disorders, including hereditary deficiencies of components of the immune system and allergic disorders, has led to the development of therapies using parenteral administration of recombinant proteins or monoclonal antibodies (1). While many of these therapies are highly effective, they are limited by the half-life of the therapeutic protein or antibody, requiring repetitive administration of days to weeks (2-15). The focus of recent work in our laboratory has been to solve this problem by substituting protein/monoclonal antibody administration with gene therapy, where current technology allows for a single administration of the gene coding for a protein or antibody to provide persistent expression of effective levels of the therapeutic protein or antibody. Gene therapy is a drug delivery platform which uses genetic material, usually in the form of coding exons of the therapeutic gene, to correct, compensate for, or prevent the development of an abnormal phenotype (16). Originally conceptualized as a strategy to treat rare hereditary disorders, gene therapy is being developed for a wide range of human disorders, including common acquired conditions (17-20). In this review, we will describe how we have adopted gene therapy technology to develop therapies for immune-related disorders, using as examples hereditary angioedema, an inherited autosomal dominant disorder, and peanut allergy, a common acquired allergic disorder.

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, Nathan's Battle Foundation, Cures Within Reach Foundation, Noah's Hope Foundation, Hope4Bridget Foundation.

In Vivo Potency Assay for Adeno-Associated Virus-Based Gene Therapy Vectors Using AAVrh.10 as an Example.

The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function. This is particularly challenging for products based on the adeno-associated virus (AAV) platform, which has poor in vitro infectivity, limiting the sensitivity and thus the usefulness of cell-based assays. A rigorous in vivo assay has been established that separately evaluates infection, transcription, and resulting protein levels with specifications for each based on real time polymerase chain reaction (DNA and RNA) and standard protein assays. For an acceptance criterion, an administered vector must have vector DNA, transgene mRNA, and transgene expressed protein each concurrently meet individual specifications or the production lot fails. Using the AAVrh.10 serotype as a model vector and three different transgenes as examples, the assay is based on intravenous administration of the vector to male mice. At 2 weeks, the harvested liver is homogenized and assessed for vector genome levels (to assess for vector delivery), mRNA (to assess vector infectivity and transcription), and protein in the liver or serum (to assess protein expression). For all AAV vectors, the assay is robust and reproducible: vector DNA (linearity 102-109 copies, coefficient of variation) intra-assay <0.8%, inter-assay <0.5%; mRNA intra-assay <3.3%, inter-assay <3.4%. The reproducibility of the assay for transgene expressed protein is product specific. This in vivo potency assay is a strategy for characterization and a quantitative lot release test, providing a path forward to meet regulatory drug requirements for any AAV gene therapy vectors.

Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH-/-). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.

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.

Peri-operative considerations in the patient with primary immune deficiency: a review.

BACKGROUND: Patients with inherited immune deficiency diseases often require surgical procedures, and their immune defects may predispose them to surgical complications. METHODS: A thorough review of pertinent literature and current practice guidelines on surgery in patients with immune deficiency. RESULTS: Peri-operative infections are a key, but not a singular, consideration in managing patients with a primary immune deficiency. Bleeding diathesis, gastrointestinal complications, pulmonary complications, and poor incision healing may also be idiosyncratic features unique to particular immune deficiency diseases. Patients with complex genetic syndromes that include immune deficiency also may display non-immunologic abnormalities that are equally important to surgical care. CONCLUSION: Greater awareness of primary immune deficiencies and a comprehensive evaluation of such patients in close consultation with an immunologist can minimize surgical complications and optimize patient outcomes.

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.

AAV-directed persistent expression of a gene encoding anti-nicotine antibody for smoking cessation.

Current strategies to help tobacco smokers quit have limited success as a result of the addictive properties of the nicotine in cigarette smoke. We hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector expressing high levels of an anti-nicotine antibody would persistently prevent nicotine from reaching its receptors in the brain. To test this hypothesis, we constructed an AAVrh.10 vector that expressed a full-length, high-affinity, anti-nicotine antibody derived from the Fab fragment of the anti-nicotine monoclonal antibody NIC9D9 (AAVantiNic). In mice treated with this vector, blood concentrations of the anti-nicotine antibody were dose-dependent, and the antibody showed high specificity and affinity for nicotine. The antibody shielded the brain from systemically administered nicotine, reducing brain nicotine concentrations to 15% of those in naïve mice. The amount of nicotine sequestered in the serum of vector-treated mice was more than seven times greater than that in untreated mice, with 83% of serum nicotine bound to immunoglobulin G. Treatment with the AAVantiNic vector blocked nicotine-mediated alterations in arterial blood pressure, heart rate, and locomotor activity. In summary, a single administration of a gene transfer vector expressing a high-affinity anti-nicotine monoclonal antibody elicited persistent (18 weeks), high titers of an anti-nicotine antibody that obviated the physiologic effects of nicotine. If this degree of efficacy translates to humans, AAVantiNic could be an effective preventative therapy for nicotine addiction.

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.

Prevalence of diverticulosis in recurrent Clostridium difficile infection.

AIM: To re-evaluate the theory that colonic diverticulosis is associated with relapse of Clostridium difficile associated disease (CDAD) in light of data suggesting increasing rates of CDAD infection and relapse. METHODS: Charts were reviewed for patients with recurrent CDAD who had also had a prior colonoscopy or flexible sigmoidoscopy. An age and gender matched control group was used to compare the prevalence of diverticulosis. RESULTS: Twenty-two patients met the study criteria, and the prevalence of diverticulosis in patients with CDAD relapse was 23% compared to 32% in age and sex matched controls (P = 0.44). A significant proportion of patients with CDAD relapse had co-morbidities associated with immune suppression. CONCLUSION: Diverticulosis does not appear to be associated with CDAD relapse.