Biomedical subjects

Ronald G Crystal

Publications and source records attributed to Ronald G Crystal.

At least 19 recordsLinked to original sources

PubMed2026

Suppression of AAV-Delivered Transgene Expression Using Artificial MicroRNAs Delivered by an Alternative AAV Serotype.

Adeno-associated virus (AAV) gene transfer vectors mediate long-term expression in nondividing cells, an advantage for treating chronic disorders. However, current platforms lack a way to selectively shut down transgene expression if adverse effects arise. To create an "off switch," we hypothesized that incorporating unique artificial microRNA (amiRNA) target sequences into an AAV expression cassette would allow subsequent suppression of transgene expression using a second AAV vector encoding the cognate amiRNA. We introduced 22-nt sequences absent from human and mouse transcriptomes into the 3' untranslated region (UTR) of a therapeutic AAV cassette. To identify optimal amiRNAs, two tandem copies of each amiRNA were cloned into the 3'UTR of an mCherry reporter gene. In vitro assessment of six amiRNA/target pairs using a dual luciferase assay identified four amiRNAs that efficiently suppressed reporter expression. Cells cotransfected with target site 3 (TS3) and amiRNA-T3B showed the greatest reduction in luciferase activity (80%, p < 0.0001) and were selected for further study. The "off-switch" system was then evaluated using an AAV5 therapeutic vector expressing a recombinant humanized anti-IgE monoclonal antibody (AAV5-TBG-anti-IgE-TS3), designed for long-term suppression of allergen-induced reactions. Co-transfection of HEK293T cells with anti-IgE-TS3 and amiRNA-T3B significantly reduced anti-IgE mRNA and protein levels relative to a control amiRNA (p < 0.0001). In vivo testing in Balb/c mice (n = 5) involved intravenous administration of AAV5-anti-IgE-TS3 (3.2 × 1010 gc), followed 4 weeks later by an AAVrh.10 amiRNA vector (AAVrh.10-TBG-amiRNA-T3B; 1 × 1011 gc). Control mice receiving only the therapeutic vector expressed 18.4 ± 13.8 µg/mL serum anti-IgE at 10 weeks. In contrast, mice receiving the amiRNA "off" vector showed marked suppression of anti-IgE (0.3 ± 0.15 µg/mL, p < 0.0001). These findings provide proof-of-concept that AAV-delivered amiRNAs can selectively switch off transgene expression, offering a strategy to improve the safety of AAV-mediated gene therapies.

Dependovirus↗

PubMed2025

Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission.

Mycobacterium tuberculosis (Mtb) must withstand physical and chemical stresses during airborne transmission, including during the desiccation of aerosols small enough to reach pulmonary alveoli in a new host. There, Mtb encounters an antimicrobial pulmonary alveolar lining fluid (ALF) before it is engulfed by macrophages. To study the genes involved in Mtb's ability to survive the transition from desiccated droplet to pulmonary alveolus in an in vitro model, we formulated a model alveolar lining fluid (MALF) that mimics the composition of ALF as inferred from human bronchoalveolar lavage fluid (BALF). We compared the transcriptome of log-phase Mtb in MALF to the transcriptome of Mtb in BALF as BALF from the lungs of healthy adults was reconstituted to compensate for the dilution of ALF by lavage (rcBALF). Mtb from log-phase culture in a standard laboratory medium survived quantitatively in MALF and rcBALF for at least 24 hours. In contrast, Mtb that had passed through earlier stages of transmission began to succumb after 3 hours in MALF, past the time when particles have been observed to be phagocytized by alveolar macrophages. Screening of a genome-wide CRISPRi library of Mtb identified 35 genes as uniquely required by Mtb to survive the transition from desiccated microdroplet into rehydration in MALF. Thirty-one of these genes are non-essential under conventional laboratory conditions and seven have unknown functions. Thirteen of the 35 genes were additionally required for Mtb to survive in macrophage-like cells cultured at the air-liquid interface with pulmonary epithelial cells. This study nominates additional members of the transmission survival genome of Mtb, illustrates that different genes may contribute to the survival of Mtb at different stages of transmission, and suggests that modeled transmission can shed light on the functions of Mtb genes whose contributions have been unknown.

Journal Article↗

PubMed2024

Suppression of CNS APOE4 Expression by miRNAs Delivered by the S2 AAVrh.10 Capsid-Modified AAV Vector.

The homozygous Apolipoprotein E (APOE4) genotype is the major risk factor for the development of early Alzheimer's disease. Genome engineering studies in mouse models of human APOE4-dependent pathology have established that reduction of APOE4 expression can rescue the phenotype. We hypothesized that APOE4 could be suppressed in the CNS of APOE4 homozygotes using adeno-associated virus (AAV) expression of microRNAs (miRNA) designed to hybridize to APOE mRNA. We screened nine different miRNAs targeting APOE following transfection in HEK293T and Huh7 cells. Optimal APOE suppression was obtained with mir2A (targeting coding region nt330-351) and mirN4 (3' untranslated region nt1142-1162). miRNA expression cassettes were designed with two copies of each of these two miRNAs co-expressed with a mCherry transgene. To optimize delivery of these miRNAs, an engineered AAVrh.10 variant was identified from a screen of multiple peptide insertions into capsid loop IV and substitutions in loop VIII. This led to identifying the AAV.S2 capsid with enhanced transduction of both neurons and glia and enhanced distribution in the brain. The engineered capsid was used to deliver the APOE miRNA suppression cassette to the hippocampus of TRE4 mice (human APOE4 knock-in replacement of the murine apoE locus). Two weeks after intra-hippocampus administration, regional expression of miRNA at the injection site was quantified at the mRNA level relative to an endogenous reference. The AAV.S2 capsid provided 2.31 ± 0.37-fold higher expression of miRNA over that provided by AAVrh.10 (p < 0.05). In the targeted region, a single intra-hippocampus AAV.S2 administration suppressed hippocampal APOE4 mRNA levels by 76.5 ± 3.9% compared with 41.3 ± 3.3% with the same cassette delivered by the wildtype AAVrh.10 capsid (p < 0.0001). We conclude that an expression cassette with two different miRNAs targeting APOE4 delivered by the AAV.S2 capsid will generate highly significant suppression of APOE4 in the CNS.

Dependovirus↗

PubMed2007

Interaction of Pneumocystis carinii with dendritic cells and resulting host responses to P. carinii.

To assess the interaction of Pneumocystis carinii with dendritic cells (DCs), and the consequences of the response of the host immune system to P. carinii antigens when DC are pulsed with P. carinii, murine DC were pulsed with P. carinii, and the resultant P. carinii host responses assessed in vitro and in vivo. P. carinii interacted with murine bone marrow-derived DC in vitro in part via mannose receptors. DC pulsed with P. carinii did not demonstrate increased expression of the cell surface markers MHC II, CD40, CD54, CD80 (B7.1), and CD86 (B7.2). The release of interleukin (IL)-4 was increased, but there was no increase in the release of interleukin (IL)-12p40, IL-10, tumor necrosis factor-alpha, IL-6, and nitrite compared with naive DC. In vivo administration of DC pulsed with P. carinii induced a P. carinii-specific response, generating CD4+ cells that proliferated and released IL-4, but not interferon-gamma, in response to P. carinii-pulsed DC in vitro. In vivo administration of DC pulsed with P. carinii also induced P. carinii-specific immunoglobulin (Ig)G1, IgG2a, and IgG2b, but not IgG3, antibodies in serum, and lung lavage fluid. Finally, CD4+ depleted mice immunized with DC pulsed with P. carinii demonstrated suppression of lung growth of P. carinii after intratracheal challenge with P. carinii at 3 and 16 weeks after immunization. These observations provide insight into DC-P. carinii interactions, and support the concept that a vaccine that includes DC pulsed with P. carinii can mount a humoral and T helper 2-type cellular response to P. carinii sufficient to suppress the growth of P. carinii in the lung.

Animals↗

PubMed2006

Enhanced survival of the LINCL mouse following CLN2 gene transfer using the rh.10 rhesus macaque-derived adeno-associated virus vector.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a lysosomal storage disorder caused by mutations in the CLN2 gene and a deficiency of tripeptidyl peptidase I (TPP-I). Prior studies with adeno-associated virus (AAV) serotype 2 or 5 mediated transfer of the CLN2 complementary DNA to the central nervous system (CNS) of CLN2(-/-) mice cleared CNS storage granules, but provided no improvement in the phenotype or survival of this model of LINCL. In this study, AAV serotypes (AAV2, AAV5, AAV8, and AAVrh.10) were compared for the delivery of the same CLN2 expression cassette. AAVrh.10, derived from rhesus macaque, provided the highest TPP-I level and maximum spread beyond the site of injection. The AAVrh.10-based vector functioned equally well in naive rats and in rats previously immunized against human serotypes of AAV. When administered to the CNS of CLN2(-/-) mice, the AAVrh.10CLN2 vector provided widespread TPP-I activity comparable to that in the wild-type mice. Importantly, the AAVrh.10CLN2-treated CLN2(-/-) mice had significant reduction in CNS storage granules and demonstrated improvement in gait, nest-making abilities, seizures, balance beam function, and grip strength, as well as having a survival advantage.

Aminopeptidases↗

PubMed2006

Responses of the human airway epithelium transcriptome to in vivo injury.

To identify genes participating in human airway epithelial repair, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed gene expression by Affymetrix microarrays with TaqMan RT-PCR confirmation. Histologically, the injured area was completely covered by a partially redifferentiated epithelial layer after 7 days; by 14 days the airway epithelium was very similar to the uninjured state. At day 7 compared with resting epithelium, there were substantial differences in gene expression pattern, with a distinctive airway epithelial "repair transcriptome" of actively proliferating cells in the process of redifferentiation. The repair transcriptome at 7 days was dominated by cell cycle, signal transduction, metabolism and transport, and transcription genes. Interestingly, the majority of differentially expressed cell cycle genes belonged to the G2 and M phases, suggesting that the proliferating cells were relatively synchronized 1 wk following injury. At 14 days postinjury, the expression profile was similar to that of resting airway epithelium. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in airway epithelial diseases.

Adult↗

PubMed2006

Up-regulation of expression of the ubiquitin carboxyl-terminal hydrolase L1 gene in human airway epithelium of cigarette smokers.

Neuroendocrine differentiation is a common feature of lung cancer and increased numbers of neuroendocrine cells and their peptides have been described in chronic smokers. To understand the effects of cigarette smoking on the gene expression profile of neuroendocrine cells, microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals [normal nonsmokers, normal smokers, smokers with early chronic obstructive lung disease (COPD), and smokers with established COPD]. Of 11 genes considered to be neuroendocrine cell specific, only ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a member of the ubiquitin proteasome pathway, was consistently up-regulated in smokers compared with nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemical analysis of bronchial biopsies of smokers compared with nonsmokers. UCHL1 expression was evident only in neuroendocrine cells of the airway epithelium in nonsmokers; however, UCHL1 was also expressed in ciliated epithelial cells in smokers. This observation may add further weight to recent observations that ciliated cells are capable of transdifferentiating to other airway epithelial cells. In the context that UCHL1 is involved in the degradation of unwanted, misfolded, or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.

Adult↗

PubMed2006

Modification of gene expression of the small airway epithelium in response to cigarette smoking.

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.

Biomarkers↗

PubMed2006

Protective immunity against respiratory tract challenge with Yersinia pestis in mice immunized with an adenovirus-based vaccine vector expressing V antigen.

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine.

Adenoviridae↗

PubMed2006

Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes.

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.

Animals↗

PubMed2006

Correction of a murine model of von Willebrand disease by gene transfer.

von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF(-/-)) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular-weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription-polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF(-/-) mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF(-/-) mice, but not control VWF(-/-) mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF(-/-) mice were less than 10% those in wild-type mice, levels of factor VIII in VWF(-/-) animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.

Animals↗

PubMed2006

Adenoviral gene transfer of stromal cell-derived factor-1 to murine tumors induces the accumulation of dendritic cells and suppresses tumor growth.

The human CXC chemokine, stromal cell-derived factor 1 (SDF-1alpha), is known to function in vitro as a chemotactic factor for lymphocytes, monocytes, and dendritic cells. In the context that dendritic cells are powerful antigen-presenting cells, we hypothesized that adenoviral gene transfer of SDF-1alpha to tumors might inhibit growth of preexisting tumors through attracting dendritic cells to the tumor. AdSDF-1alpha mediated the expression of SDF-1alpha mRNA and protein in A549 cells in vitro, and the supernatant of the AdSDF-1alpha-infected A549 cells showed chemotactic activity for dendritic cells. When syngeneic murine CT26 colon carcinoma tumors (BALB/c) and B16 melanoma and Lewis lung cell carcinoma (C57Bl/6) were injected with AdSDF-1alpha (5 x 10(8) plaque-forming units), there was an accumulation of dendritic cells and CD8(+) cells within the tumor and significant inhibition of tumor growth compared with tumors injected with PBS or AdNull (control vector). The injection of AdSDF-1alpha into tumors induced the inflammatory enlargement and the accumulation of dendritic cells in the draining lymph node. Intratumoral AdSDF-1alpha administration elicited tumor-specific CTLs and adoptive transfer of splenocytes from AdSDF-1alpha-treated mice resulted in the elongation of survival after tumor challenge. Interestingly, in wild-type and CD4(-/-) mice but not in CD8(-/-) mice, AdSDF-1alpha inhibited the growth of the tumor. These observations suggest that adenoviral gene transfer of SDF-1alpha may be a useful strategy to accumulate dendritic cells in tumors and evoke antitumor immune responses to inhibit tumor growth.

Adenocarcinoma↗

PubMed2006

Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking.

Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, approximately 20 pack-year smokers compared to healthy nonsmokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to nonsmokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related function, signal transduction, and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in AM in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of AM associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.

Computer Systems↗

PubMed2006

Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro.

Bone morphogenic proteins (BMPs) are growth factors important for skeletal development and bone growth. Noggin, one of the soluble BMP antagonists, regulates the action of BMPs on mesenchymal precursor cells, partially through a feedback type of inhibition. In this study, we constructed a novel BMP2/7 'fusion gene' that encodes both BMP2 and BMP7 genes in tandem by a linker. Polymerase chain reaction (PCR) and Western blotting showed that the BMP2/7 fusion gene construct led to the production of BMP2/7 heterodimers in A549 'producer' cells. When applied to C2C12 myoblastic cells, BMP2/7 heterodimers increased alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression (markers of osteoblastic differentiation) more effectively than either BMP2 or BMP7 homodimers. Moreover, this heterodimer induced significantly lower levels of Noggin expression in C2C12 cells than respective homodimers at similar doses. The addition of Noggin did not affect the heterodimer's activities in increasing osteoblastic differentiation in C2C12 cells. In contrast, BMP2 and BMP7 homodimers were largely inhibited by Noggin. Our finding suggests that the 'fusion gene' construct led to the production of bioactive BMP2/7 heterodimers, which were not antagonized by Noggin as effectively as it to BMP homodimers. The weaker Noggin antagonism on BMP heterodimers compared to homodimers may contribute to increased osteogenic potency of heterodimers in vitro and in vivo.

Alkaline Phosphatase↗

PubMed2006

Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2(-/-) mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2(+/+) control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Aminopeptidases↗

PubMed2006

Genetic medicines: treatment strategies for hereditary disorders.

The treatment of the more than 1,800 known monogenic hereditary disorders will depend on the development of 'genetic medicines' - therapies that use the transfer of DNA and/or RNA to modify gene expression to correct or compensate for an abnormal phenotype. Strategies include the use of somatic stem cells, gene transfer, RNA modification and, in the future, embryonic stem cells. Despite the efficacy of these technologies in treating experimental models of hereditary disorders, applying them successfully in the clinic is a great challenge, which will only be overcome by expending considerable intellectual and economic resources, and by solving societal concerns about modifications of the human genetic repertoire.

Gene Transfer Techniques↗

PubMed2006

A common mechanism for cytoplasmic dynein-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes.

During infection, adenovirus-associated virus (AAV) undergoes microtubule-dependent retrograde transport as part of a program of vectorial transport of viral genome to the nucleus. A microtubule binding assay was used to evaluate the hypothesis that cytoplasmic dynein mediates AAV interaction with microtubules. Binding of AAV serotype 2 (AAV2) was enhanced in a nucleotide-dependent manner by the presence of total cellular microtubule-associated proteins (MAPs) but not cytoplasmic dynein-depleted MAPs. Excess AAV2 capsid protein prevented microtubule binding by AAV serotypes 2, 5, and rh.10, as well as adenovirus serotype 5, indicating that similar binding sites are used by these viruses.

Adenoviridae↗

PubMed2006

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

Adenoviridae↗