Browsing by Subject "Transgenes"

Now showing 1 - 19 of 19
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    Antibody formation and mannose-6-phosphate receptor expression impact the efficacy of muscle-specific transgene expression in murine Pompe disease.
    (J Gene Med, 2010-11) Sun, Baodong; Li, Songtao; Bird, Andrew; Yi, Haiqing; Kemper, Alex; Thurberg, Beth L; Koeberl, Dwight D
    BACKGROUND: Lysosomal storage disorders such as Pompe disease can be more effectively treated, if immune tolerance to enzyme or gene replacement therapy can be achieved. Alternatively, immune responses against acid α-glucosidase (GAA) might be evaded in Pompe disease through muscle-specific expression of GAA with adeno-associated virus (AAV) vectors. METHODS: An AAV vector containing the MHCK7 regulatory cassette to drive muscle-specific GAA expression was administered to GAA knockout (KO) mice, immune tolerant GAA-KO mice and mannose-6-phosphate deficient GAA-KO mice. GAA activity and glycogen content were analyzed in striated muscle to determine biochemical efficacy. RESULTS: The biochemical efficacy from GAA expression was slightly reduced in GAA-KO mice, as demonstrated by higher residual glycogen content in skeletal muscles. Next, immune tolerance to GAA was induced in GAA-KO mice by co-administration of a second AAV vector encoding liver-specific GAA along with the AAV vector encoding muscle-specific GAA. Antibody formation was prevented by liver-specific GAA, and the biochemical efficacy of GAA expression was improved in the absence of antibodies, as demonstrated by significantly reduced glycogen content in the diaphragm. Efficacy was reduced in old GAA-KO mice despite the absence of antibodies. The greatest impact upon gene therapy was observed in GAA-KO mice lacking the mannose-6-phosphate receptor in muscle. The clearance of stored glycogen was markedly impaired despite high GAA expression in receptor-deficient Pompe disease mice. CONCLUSIONS: Overall, antibody formation had a subtle effect upon efficacy, whereas the absence of mannose-6-phosphate receptors markedly impaired muscle-targeted gene therapy in murine Pompe disease.
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    Calcineurin activation causes retinal ganglion cell degeneration.
    (Mol Vis, 2012) Qu, Juan; Matsouaka, Roland; Betensky, Rebecca A; Hyman, Bradley T; Grosskreutz, Cynthia L
    PURPOSE: We previously reported that calcineurin, a Ca(2+)/calmodulin-dependent serine/threonine phosphatase, is activated and proposed that it participates in retinal ganglion cell (RGC) apoptosis in two rodent ocular hypertension models. In this study, we tested whether calcineurin activation by itself, even in the absence of ocular hypertension, is sufficient to cause RGC degeneration. METHODS: We compared RGC and optic nerve morphology after adeno-associated virus serotype 2 (AAV2)-mediated transduction of RGCs with constitutively active calcineurin (CaNCA) or unactivated, wild-type calcineurin (CaNwt). Retinas and optic nerves were harvested 7-16 weeks after injection of the AAV into mouse vitreous. In flatmounted retinas, the transduced RGCs were identified with immunohistochemistry. The morphology of the RGCs was revealed by immunostaining for neurofilament SMI32 or by using GFP-M transgenic mice. A modified Sholl analysis was applied to analyze the RGC dendritic morphology. Optic nerve damage was assessed with optic nerve grading according to the Morrison standard. RESULTS: CaNwt and CaNCA were highly expressed in the injected eyes. Compared to the CaNwt-expressing RGCs, the CaNCA-expressing RGCs had smaller somas, smaller dendritic field areas, shorter total dendrite lengths, and simpler dendritic branching patterns. At 16 weeks, the CaNCA-expressing eyes had greater optic nerve damage than the CaNwt-expressing eyes. CONCLUSIONS: Calcineurin activation is sufficient to cause RGC dendritic degeneration and optic nerve damage. These data support the hypothesis that calcineurin activation is an important mediator of RGC degeneration, and are consistent with the hypothesis that calcineurin activation may contribute to RGC neurodegeneration in glaucoma.
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    Costimulation of adenylyl cyclase and phospholipase C by a mutant alpha 1B-adrenergic receptor transgene promotes malignant transformation of thyroid follicular cells.
    (Endocrinology, 1997-01) Ledent, C; Denef, JF; Cottecchia, S; Lefkowitz, R; Dumont, J; Vassart, G; Parmentier, M
    Proliferation of thyroid follicular cells is controlled by three intra-cellular cascades [cAMP, inositol 1,4,5-triphosphate (IP3)/Ca2+/diacylglycerol (DAG), and tyrosine kinases] that are activated by distinct extracellular signals and receptors. We had previously generated a transgenic mouse model in which the cAMP cascade was permanently stimulated in thyroid cells by an adenosine A2a receptor (Tg-A2aR model). In the present work, we have generated a transgenic model characterized by the chronic stimulation of both adenylyl cyclase and phospholipase C in thyroid follicular cells. The bovine thyroglobulin gene promoter was used to direct the expression of a constitutively active mutant of the alpha 1B adrenergic receptor, which is known to couple to both cascades in transfected cell lines. The expression of the transgene resulted, as expected, in the activation of phospholipase C and adenylyl cyclase, as demonstrated by the direct measurement of IP3 and cAMP in thyroid tissue. The phenotype resulting from this dual stimulation included growth stimulation, hyperfunction, cell degeneracy attributed to the overproduction of free radicals, and the development of malignant nodules invading the capsule, muscles, and blood vessels. Differentiated metastases were found occasionally in old animals. The development of malignant lesions was more frequent and of earlier onset than in our previous Tg-A2aR model, in which only the cAMP cascade was stimulated. These observations demonstrate that the cAMP and IP3/Ca2+/DAG cascades can cooperate in vivo toward the development of thyroid follicular cell malignancies.
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    Facilitative glucose transporter Glut1 is actively excluded from rod outer segments.
    (J Cell Sci, 2010-11-01) Gospe, Sidney M; Baker, Sheila A; Arshavsky, Vadim Y
    Photoreceptors are among the most metabolically active cells in the body, relying on both oxidative phosphorylation and glycolysis to satisfy their high energy needs. Local glycolysis is thought to be particularly crucial in supporting the function of the photoreceptor's light-sensitive outer segment compartment, which is devoid of mitochondria. Accordingly, it has been commonly accepted that the facilitative glucose transporter Glut1 responsible for glucose entry into photoreceptors is localized in part to the outer segment plasma membrane. However, we now demonstrate that Glut1 is entirely absent from the rod outer segment and is actively excluded from this compartment by targeting information present in its cytosolic C-terminal tail. Our data indicate that glucose metabolized in the outer segment must first enter through other parts of the photoreceptor cell. Consequently, the entire energy supply of the outer segment is dependent on diffusion of energy-rich substrates through the thin connecting cilium that links this compartment to the rest of the cell.
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    G protein signaling and vein graft intimal hyperplasia: reduction of intimal hyperplasia in vein grafts by a Gbetagamma inhibitor suggests a major role of G protein signaling in lesion development.
    (Arterioscler Thromb Vasc Biol, 1998-08) Davies, MG; Fulton, Gregory J; Hagen, Per-Otto Frode; Huynh, Tam; Koch, Walter J; Lefkowitz, Robert J; Svendsen, E
    Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide-binding (G) protein expression and function. Several serum mitogens that act through G protein-coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein betagamma subunit (Gbetagamma)-mediated activation of p21ras. This study examines the role of Gbetagamma signaling in intimal hyperplasia by targeting a gene encoding a specific Gbetagamma inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK(CT)), contains a Gbetagamma-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by betaARK(CT) treatment. Thus, it appears that Gbetagamma-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gbetagamma signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.
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    Gene therapy for glycogen storage diseases.
    (Human molecular genetics, 2019-10) Kishnani, Priya S; Sun, Baodong; Koeberl, Dwight D
    The focus of this review is the development of gene therapy for glycogen storage diseases (GSDs). GSD results from the deficiency of specific enzymes involved in the storage and retrieval of glucose in the body. Broadly, GSDs can be divided into types that affect liver or muscle or both tissues. For example, glucose-6-phosphatase (G6Pase) deficiency in GSD type Ia (GSD Ia) affects primarily the liver and kidney, while acid α-glucosidase (GAA) deficiency in GSD II causes primarily muscle disease. The lack of specific therapy for the GSDs has driven efforts to develop new therapies for these conditions. Gene therapy needs to replace deficient enzymes in target tissues, which has guided the planning of gene therapy experiments. Gene therapy with adeno-associated virus (AAV) vectors has demonstrated appropriate tropism for target tissues, including the liver, heart and skeletal muscle in animal models for GSD. AAV vectors transduced liver and kidney in GSD Ia and striated muscle in GSD II mice to replace the deficient enzyme in each disease. Gene therapy has been advanced to early phase clinical trials for the replacement of G6Pase in GSD Ia and GAA in GSD II (Pompe disease). Other GSDs have been treated in proof-of-concept studies, including GSD III, IV and V. The future of gene therapy appears promising for the GSDs, promising to provide more efficacious therapy for these disorders in the foreseeable future.
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    Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.
    (Circ Res, 2000-01-07) Eckhart, AD; Duncan, SJ; Penn, RB; Benovic, JL; Lefkowitz, RJ; Koch, WJ
    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.
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    Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine pompe disease.
    (Hum Gene Ther, 2012-05) Zhang, P; Sun, B; Osada, T; Rodriguiz, R; Yang, XY; Luo, X; Kemper, AR; Clay, TM; Koeberl, DD
    Pompe disease can be treated effectively, if immune tolerance to enzyme replacement therapy (ERT) with acid α-glucosidase (GAA) is present. An adeno-associated viral (AAV) vector carrying a liver-specific regulatory cassette to drive GAA expression (AAV-LSPhGAA) established immune tolerance in GAA knockout (KO) mice, whereas ubiquitous expression with AAV-CBhGAA provoked immune responses. Therefore, we investigated the hypothesis that immune tolerance induced by hepatic-restricted expression was dominant. AAV-LSPhGAA and AAV-CBhGAA were administered singly or in combination to groups of adult GAA-KO mice, and AAV-LSPhGAA induced immune tolerance even in combination with AAV-CBhGAA. The dual vector approach to GAA expression improved biochemical correction of GAA deficiency and glycogen accumulations at 18 weeks, and improved motor function testing including wire-hang and grip-strength testing. The greatest efficacy was demonstrated by dual vector administration, when both vectors were pseudotyped as AAV8. T cells from mice injected with AAV-LSPhGAA failed to proliferate at all after an immune challenge with GAA and adjuvant, whereas mock-treated GAA-KO mice mounted vigorous T cell proliferation. Unlike AAV-LSPhGAA, AAV-CBhGAA induced selective cytokine and chemokine expression in liver and spleen after the immune challenge. AAV-CBhGAA transduced dendritic cells and expressed high-level GAA, whereas AAV-LSPhGAA failed to express GAA in dendritic cells. The level of transduction in liver was much higher after dual AAV8 vector administration at 18 weeks, in comparison with either vector alone. Dual vector administration failed to provoke antibody formation in response to GAA expression with AAV-CBhGAA; however, hepatic-restricted expression from dual vector expression did not prevent antibody formation after a strong immune challenge with GAA and adjuvant. The relevance of immune tolerance to gene therapy in Pompe disease indicates that hepatic expression might best be combined with nonhepatic expression, achieving the benefits of ubiquitous expression in addition to evading deleterious immune responses.
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    In vivo inhibition of elevated myocardial beta-adrenergic receptor kinase activity in hybrid transgenic mice restores normal beta-adrenergic signaling and function.
    (Circulation, 1999-08-10) Akhter, SA; Eckhart, AD; Rockman, HA; Shotwell, K; Lefkowitz, RJ; Koch, WJ
    BACKGROUND: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function. METHODS AND RESULTS: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice. CONCLUSIONS: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.
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    In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.
    (Circulation, 2001-03-06) Shah, AS; White, DC; Emani, S; Kypson, AP; Lilly, RE; Wilson, K; Glower, DD; Lefkowitz, RJ; Koch, WJ
    BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.
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    Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.
    (Molecular therapy : the journal of the American Society of Gene Therapy, 2020-06) Miyadera, Keiko; Conatser, Laura; Llanga, Telmo A; Carlin, Kendall; O'Donnell, Patricia; Bagel, Jessica; Song, Liujiang; Kurtzberg, Joanne; Samulski, R Jude; Gilger, Brian; Hirsch, Matthew L
    Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.
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    Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.
    (J Clin Invest, 1997-01-15) Drazner, MH; Peppel, KC; Dyer, S; Grant, AO; Koch, WJ; Lefkowitz, RJ
    Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.
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    Preclinical Development of New Therapy for Glycogen Storage Diseases.
    (Curr Gene Ther, 2015) Sun, Baodong; Brooks, Elizabeth D; Koeberl, Dwight D
    Glycogen storage disease (GSD) consists of more than 10 discrete conditions for which the biochemical and genetic bases have been determined, and new therapies have been under development for several of these conditions. Gene therapy research has generated proof-of-concept for GSD types I (von Gierke disease) and II (Pompe disease). Key features of these gene therapy strategies include the choice of vector and regulatory cassette, and recently adeno-associated virus (AAV) vectors containing tissue-specific promoters have achieved a high degree of efficacy. Efficacy of gene therapy for Pompe disease depend upon the induction of immune tolerance to the therapeutic enzyme. Efficacy of von Gierke disease is transient, waning gradually over the months following vector administration. Small molecule therapies have been evaluated with the goal of improving standard of care therapy or ameliorating the cellular abnormalities associated with specific GSDs. The receptor-mediated uptake of the therapeutic enzyme in Pompe disease was enhanced by administration of β2 agonists. Rapamycin reduced the liver fibrosis observed in GSD III. Further development of gene therapy could provide curative therapy for patients with GSD, if efficacy from preclinical research is observed in future clinical trials and these treatments become clinically available.
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    Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection.
    (PLoS One, 2015) Bráz, João M; Wang, Fan; Basbaum, Allan I
    Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Cre-expressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Cre-dependent pseudorabies or rabies vectors, which results in labeling of poly- and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat.
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    Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.
    (Stem cell reviews and reports, 2015-08) Zhou, Hongyan; Martinez, Hector; Sun, Bruce; Li, Aiqun; Zimmer, Matthew; Katsanis, Nicholas; Davis, Erica E; Kurtzberg, Joanne; Lipnick, Scott; Noggle, Scott; Rao, Mahendra; Chang, Stephen
    Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.
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    Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-xL transgenic mice.
    (J Exp Med, 1999-08-02) Takahashi, Y; Cerasoli, DM; Dal Porto, JM; Shimoda, M; Freund, R; Fang, W; Telander, DG; Malvey, EN; Mueller, DL; Behrens, TW; Kelsoe, G
    The role of apoptosis in affinity maturation was investigated by determining the affinity of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific antibody-forming cells (AFCs) and serum antibody in transgenic mice that overexpress a suppressor of apoptosis, Bcl-xL, in the B cell compartment. Although transgenic animals briefly expressed higher numbers of splenic AFCs after immunization, the bcl-xL transgene did not increase the number or size of germinal centers (GCs), alter the levels of serum antibody, or change the frequency of NP-specific, long-lived AFCs. Nonetheless, the bcl-xL transgene product, in addition to endogenous Bcl-xL, reduced apoptosis in GC B cells and resulted in the expansion of B lymphocytes bearing VDJ rearrangements that are usually rare in primary anti-NP responses. Long-lived AFCs bearing these noncanonical rearrangements were frequent in the bone marrow and secreted immunoglobulin G(1) antibodies with low affinity for NP. The abundance of noncanonical cells lowered the average affinity of long-lived AFCs and serum antibody, demonstrating that Bcl-xL and apoptosis influence clonal selection/maintenance for affinity maturation.
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    Restoration of beta-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer.
    (Proc Natl Acad Sci U S A, 1997-10-28) Akhter, SA; Skaer, CA; Kypson, AP; McDonald, PH; Peppel, KC; Glower, DD; Lefkowitz, RJ; Koch, WJ
    Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring beta-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular beta-adrenergic signaling defects including down-regulation of myocardial beta-adrenergic receptors (beta-ARs), functional beta-AR uncoupling, and an up-regulation of the beta-AR kinase (betaARK1). Adenoviral-mediated gene transfer of the human beta2-AR or an inhibitor of betaARK1 to these failing myocytes led to the restoration of beta-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of betaARK1 activity in the heart.
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    The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.
    (PLoS One, 2015) Walton, Eric M; Cronan, Mark R; Beerman, Rebecca W; Tobin, David M
    Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.
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    The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia.
    (Mol Genet Metab, 2013-11) Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D; Mansfield, Brian C; Chou, Janice Y
    Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.