Dorsal root ganglion (DRG) neuron dysfunction occurs in a variety of sensory neuronopathies for which there are currently no satisfactory treatments. Here we describe the development of a strategy to target therapeutic genes to DRG neurons for the treatment of these disorders. We genetically modified an adenovirus (Ad) to generate a helper virus (HV) that was detargeted for native adenoviral tropism and contained DRG homing peptides in the adenoviral capsid fiber protein; we used this HV to generate DRG-targeted helper-dependent Ad (HDAd). In mice, intrathecal injection of this HDAd produced a 100-fold higher transduction of DRG neurons and a markedly attenuated inflammatory response compared with unmodified HDAd. We also injected HDAd encoding the β subunit of β-hexosaminidase (Hexb) into Hexb-deficient mice, a model of the neuronopathy Sandhoff disease. Delivery of the DRG-targeted HDAd reinstated neuron-specific Hexb production, reversed gangliosidosis, and ameliorated peripheral sensory dysfunction. The development of DRG neuron–targeted HDAd with proven efficacy in a preclinical model may have implications for the treatment of sensory neuronopathies of diverse etiologies.
Tomoya Terashima, Kazuhiro Oka, Angelika B. Kritz, Hideto Kojima, Andrew H. Baker, Lawrence Chan
Liver sinusoidal endothelial cells are a major endogenous source of Factor VIII (FVIII), lack of which causes the human congenital bleeding disorder hemophilia A. Despite extensive efforts, gene therapy using viral vectors has shown little success in clinical hemophilia trials. Here we achieved cell type–specific gene targeting using hyaluronan- and asialoorosomucoid-coated nanocapsules, generated using dispersion atomization, to direct genes to liver sinusoidal endothelial cells and hepatocytes, respectively. To highlight the therapeutic potential of this approach, we encapsulated Sleeping Beauty transposon expressing the B domain–deleted canine FVIII in cis with Sleeping Beauty transposase in hyaluronan nanocapsules and injected them intravenously into hemophilia A mice. The treated mice exhibited activated partial thromboplastin times that were comparable to those of wild-type mice at 5 and 50 weeks and substantially shorter than those of untreated controls at the same time points. Further, plasma FVIII activity in the treated hemophilia A mice was nearly identical to that in wild-type mice through 50 weeks, while untreated hemophilia A mice exhibited no detectable FVIII activity. Thus, Sleeping Beauty transposon targeted to liver sinusoidal endothelial cells provided long-term expression of FVIII, without apparent antibody formation, and improved the phenotype of hemophilia A mice.
Betsy T. Kren, Gretchen M. Unger, Lucas Sjeklocha, Alycia A. Trossen, Vicci Korman, Brenda M. Diethelm-Okita, Mark T. Reding, Clifford J. Steer
Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid–derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.
Gary C. Pien, Etiena Basner-Tschakarjan, Daniel J. Hui, Ashley N. Mentlik, Jonathan D. Finn, Nicole C. Hasbrouck, Shangzhen Zhou, Samuel L. Murphy, Marcela V. Maus, Federico Mingozzi, Jordan S. Orange, Katherine A. High
Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C.
Andrew C. Edmondson, Robert J. Brown, Sekar Kathiresan, L. Adrienne Cupples, Serkalem Demissie, Alisa Knodle Manning, Majken K. Jensen, Eric B. Rimm, Jian Wang, Amrith Rodrigues, Vaneeta Bamba, Sumeet A. Khetarpal, Megan L. Wolfe, Stephanie DerOhannessian, Mingyao Li, Muredach P. Reilly, Jens Aberle, David Evans, Robert A. Hegele, Daniel J. Rader
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3′ untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a–specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell–based therapies, including cancer immunotherapy.
Eirini P. Papapetrou, Damian Kovalovsky, Laurent Beloeil, Derek Sant’Angelo, Michel Sadelain
Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.
Stefan Bröer, Charles G. Bailey, Sonja Kowalczuk, Cynthia Ng, Jessica M. Vanslambrouck, Helen Rodgers, Christiane Auray-Blais, Juleen A. Cavanaugh, Angelika Bröer, John E.J. Rasko
Bitter taste–sensing G protein–coupled receptors (type 2 taste receptors [T2Rs]) are expressed in taste receptor cells of the tongue, where they play an important role in limiting ingestion of bitter-tasting, potentially toxic compounds. T2Rs are also expressed in gut-derived enteroendocrine cells, where they have also been hypothesized to play a role in limiting toxin absorption. In this study, we have shown that T2R gene expression in both cultured mouse enteroendocrine cells and mouse intestine is regulated by the cholesterol-sensitive SREBP-2. In addition, T2R stimulation of cholecystokinin (CCK) secretion was enhanced directly by SREBP-2 in cultured cells and in mice fed chow supplemented with lovastatin and ezetimibe (L/E) to decrease dietary sterol absorption and increase nuclear activity of SREBP-2. Low-cholesterol diets are naturally composed of high amounts of plant matter that is likely to contain dietary toxins, and CCK is known to improve dietary absorption of fats, slow gastric emptying, and decrease food intake. Thus, these studies suggest that SREBP-2 activation of bitter signaling receptors in the intestine may sensitize the gut to a low-fat diet and to potential accompanying food-borne toxins that make it past the initial aversive response in the mouth.
Tae-Il Jeon, Bing Zhu, Jarrod L. Larson, Timothy F. Osborne
Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.
Shao H. Yang, Douglas A. Andres, H. Peter Spielmann, Stephen G. Young, Loren G. Fong
Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin–Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies.
Alberto J. Schuhmacher, Carmen Guerra, Vincent Sauzeau, Marta Cañamero, Xosé R. Bustelo, Mariano Barbacid
Cytosolic phospholipase A2α (cPLA2α) hydrolyzes arachidonic acid from cellular membrane phospholipids, thereby providing enzymatic substrates for the synthesis of eicosanoids, such as prostaglandins and leukotrienes. Considerable understanding of cPLA2α function has been derived from investigations of the enzyme and from cPLA2α-null mice, but knowledge of discrete roles for this enzyme in humans is limited. We investigated a patient hypothesized to have an inherited prostanoid biosynthesis deficiency due to his multiple, complicated small intestinal ulcers despite no use of cyclooxygenase inhibitors. Levels of thromboxane B2 and 12-hydroxyeicosatetraenoic acid produced by platelets and leukotriene B4 released from calcium ionophore–activated blood were markedly reduced, indicating defective enzymatic release of the arachidonic acid substrate for the corresponding cyclooxygenase and lipoxygenases. Platelet aggregation and degranulation induced by adenosine diphosphate or collagen were diminished but were normal in response to arachidonic acid. Two heterozygous single base pair mutations and a known SNP were found in the coding regions of the patient’s cPLA2α genes (p.[Ser111Pro]+[Arg485His; Lys651Arg]). The total PLA2 activity in sonicated platelets was diminished, and the urinary metabolites of prostacyclin, prostaglandin E2, prostaglandin D2, and thromboxane A2 were also reduced. These findings characterize what we believe is a novel inherited deficiency of cPLA2.
David H. Adler, Joy D. Cogan, John A. Phillips III, Nathalie Schnetz-Boutaud, Ginger L. Milne, Tina Iverson, Jeffrey A. Stein, David A. Brenner, Jason D. Morrow, Olivier Boutaud, John A. Oates