Allison M. Keeler

Lack of Cystic Fibrosis Transmembrane Conductance Regulator in CD3+ Lymphocytes Leads to Aberrant Cytokine Secretion and Hyperinflammatory Adaptive Immune Responses

Cystic fibrosis (CF), the most common fatal monogenic disease in the United States, results from mutations in CF transmembrane conductance regulator (CFTR), a chloride channel. The mechanisms by which CFTR mutations cause lung disease in CF are not fully defined but may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways. We have shown that Cftr(-/-) mice mount an exaggerated IgE response toward Aspergillus fumigatus, with higher levels of IL-13 and IL-4, mimicking both the T helper cell type 2-biased immune responses seen in patients with CF. Herein, we demonstrate that these aberrations are primarily due to Cftr deficiency in lymphocytes rather than in the epithelium. Adoptive transfer experiments with CF splenocytes confer a higher IgE response to Aspergillus fumigatus compared with hosts receiving wild-type splenocytes. The predilection of Cftr-deficient lymphocytes to mount T helper cell type 2 responses with high IL-13 and IL-4 was confirmed by in vitro antigen recall experiments. Conclusive data on this phenomenon were obtained with conditional Cftr knockout mice, where mice lacking Cftr in T cell lineages developed higher IgE than their wild-type control littermates. Further analysis of Cftr-deficient lymphocytes revealed an enhanced intracellular Ca(2+) flux in response to T cell receptor activation. This was accompanied by an increase in nuclear localization of the calcium-sensitive transcription factor, nuclear factor of activated T cell, which could drive the IL-13 response. In summary, our data identified that CFTR dysfunction in T cells can lead directly to aberrant immune responses. These findings implicate the lymphocyte population as a potentially important target for CF therapeutics.

Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

Systemic AAV9 gene transfer in adult GM1 gangliosidosis mice reduces lysosomal storage in CNS and extends lifespan

GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease where GLB1 gene mutations result in a reduction or absence of lysosomal acid β-galactosidase (βgal) activity. βgal deficiency leads to accumulation of GM1-ganglioside in the central nervous system (CNS). GM1 is characterized by progressive neurological decline resulting in generalized paralysis, extreme emaciation and death. In this study, we assessed the therapeutic efficacy of an adeno-associated virus (AAV) 9-mβgal vector infused systemically in adult GM1 mice (βGal(-/-)) at 1 × 10(11) or 3 × 10(11) vector genomes (vg). Biochemical analysis of AAV9-treated GM1 mice showed high βGal activity in liver and serum. Moderate βGal levels throughout CNS resulted in a 36-76% reduction in GM1-ganglioside content in the brain and 75-86% in the spinal cord. Histological analyses of the CNS of animals treated with 3 × 10(11) vg dose revealed increased presence of βgal and clearance of lysosomal storage throughout cortex, hippocampus, brainstem and spinal cord. Storage reduction in these regions was accompanied by a marked decrease in astrogliosis. AAV9 treatment resulted in improved performance in multiple tests of motor function and behavior. Also the majority of GM1 mice in the 3 × 10(11) vg cohort retained ambulation and rearing despite reaching the humane endpoint due to weight loss. Importantly, the median survival of AAV9 treatment groups (316-576 days) was significantly increased over controls (250-264 days). This study shows that moderate widespread expression of βgal in the CNS of GM1 gangliosidosis mice is sufficient to achieve significant biochemical impact with phenotypic amelioration and extension in lifespan.

Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 1012 vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD−/− mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD−/− mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD−/− mice maintained euglycemia, whereas untreated VLCAD−/− mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

Cell and gene therapy for genetic diseases: inherited disorders affecting the lung and those mimicking sudden infant death syndrome.

Some of the first human gene therapy trials targeted diseases of the lung and provided important information that will continue to help shape future trials. Here we describe both cell and gene therapies for lung diseases such as cystic fibrosis and alpha-1 antitrypsin disorder as well as fatty acid oxidation disorders that mimic sudden infant death syndrome (SIDS). Human clinical gene therapy trials for cystic fibrosis and alpha-1 antitrypsin have been performed using a variety of vectors including adenovirus, adeno-associated virus, and nonviral vectors. No human clinical gene therapy trials have been performed for disorders of fatty acid oxidation; however, important proof-of-principle studies have been completed for multiple fatty acid oxidation disorders. Important achievements have been made and have yet to come for cell and gene therapies for disorders of the lung and those mimicking SIDS.

CAR T-Cell Therapy: Progress and Prospects

Lentivirus-mediated transduction of autologous T cells with a chimeric antigen receptor (CAR) to confer a desired epitope specificity as a targeted immunotherapy for cancer has been among the first human gene therapy techniques to demonstrate widespread therapeutic efficacy. Other approaches to using gene therapy to enhance antitumor immunity have been less specific and less effective. These have included amplification, marking, and cytokine transduction of tumor infiltrating lymphocytes, recombinant virus-based expression of tumor antigens as a tumor vaccine, and transduction of antigen-presenting cells with tumor antigens. Unlike any of those methods, the engineering of CAR T cells combine specific monoclonal antibody gene sequences to confer epitope specificity and other T-cell receptor and activation domains to create a self-contained single vector approach to produce a very specific antitumor response, as is seen with CD19-directed CAR T cells used to treat CD19-expressing B-cell malignancies. Recent success with these therapies is the culmination of a long step-wise iterative process of improvement in the design of CAR vectors. This review aims to summarize this long series of advances in the development of effective CAR vector since their initial development in the 1990s, and to describe emerging approaches to design that promise to enhance and widen the human gene therapy relevance of CAR T-cell therapy in the future.

Modulation of Exaggerated-IgE Allergic Responses by Gene Transfer-mediated Antagonism of IL-13 and IL-17e.

Asthma and allergic rhinitis are almost invariable accompanied by elevated levels of immunoglobin E (IgE), and more importantly a genetic link between IgE levels and airway hyper-responsiveness has been established. We hypothesized that expression of soluble receptors directed against interleukin (IL)-13 and IL-17e would prevent the cytokines from engaging the cell-bound receptors and therefore help to attenuate allergic responses in a Cftr(-/-)-dependent mouse model of exaggerated-IgE responses. Cftr(-/-) mice were injected with recombinant adeno-associated virus 1 (rAAV1) intramuscularly expressing soluble receptors to IL-17e (IL-17Rh1fc) or IL-13 (IL-13Ralpha2Fc). Total IgE levels, in mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapy, were lower than in the control group. Interestingly Aspergillus fumigatus (Af)-specific IgE levels were undetectable in both the mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapies. Further flow cytometry analysis of intracellular gene expression suggests that blocking IL-17e may be interfering with signaling upstream of CD4+ and CD11b+ cells and reducing IgE levels by affecting signaling on these cell populations. In contrast it appears that IL-13 blockade acts downstream to reduce IgE levels probably by directly affecting B-cell maturation. These studies demonstrate the feasibility of targeting T helper 2 (Th2) cytokines with rAAV-delivered fusion proteins as a means to treat aberrant immune responses.

Systemic Delivery of AAVB1-GAA Clears Glycogen and Prolongs Survival in a Mouse Model of Pompe Disease

Pompe disease is an autosomal recessive glycogen storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption in muscle and the central nervous system (CNS). Adeno-associated virus (AAV) gene therapy is ideal for Pompe disease, since a single systemic injection may correct both muscle and CNS pathologies. Using the Pompe mouse (B6;129-GaaTm1Rabn/J), this study sought to explore if AAVB1, a newly engineered vector with a high affinity for muscle and CNS, reduces systemic weakness and improves survival in adult mice. Three-month-old Gaa-/- animals were injected with either AAVB1 or AAV9 vectors expressing GAA and tissues were harvested 6 months later. Both AAV vectors prolonged survival. AAVB1-treated animals had a robust weight gain compared to the AAV9-treated group. Vector genome levels, GAA enzyme activity, and histological analysis indicated that both vectors transduced the heart efficiently, leading to glycogen clearance, and transduced the diaphragm and CNS at comparable levels. AAVB1-treated mice had higher GAA activity and greater glycogen clearance in the tongue. Finally, AAVB1-treated animals showed improved respiratory function comparable to wild-type animals. In conclusion, AAVB1-GAA offers a promising therapeutic option for the treatment of muscle and CNS in Pompe disease.

Restrictive Lung Disease in the Cu/Zn Superoxide-Dismutase 1 G93A Amyotrophic Lateral Sclerosis Mouse Model

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Airway smooth muscle dysfunction in Pompe (Gaa−/−) mice

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons, and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease, the Gaa-/- mouse, we compared histology, pulmonary mechanics, airway smooth muscle (ASM) function, and calcium signaling between Gaa-/- and age-matched wild-type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (MCh) and potassium chloride (KCl) and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi and impairs the ability of lower ASM to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, ASM dysfunction may contribute to respiratory impairments in Pompe disease.