Gene Therapy

Tang JCY, Drokhlyansky E, Etemad B, Rudolph S, Guo B, Wang S, Ellis EG, Li JZ, Cepko CL. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. Elife 2016;5Abstract

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular proteins.

Garanto A, Chung DC, Duijkers L, Corral-Serrano JC, Messchaert M, Xiao R, Bennett J, Vandenberghe LH, Collin RWJ. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet 2016;Abstract

Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991+1655A>G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential treatment of naked and AAV-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.

Beier KT, Mundell NA, Pan AY, Cepko CL. Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors. Curr Protoc Neurosci 2016;74:1.26.1-1.26.27.Abstract

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies. © 2016 by John Wiley & Sons, Inc.

Mayer C, Jaglin XH, Cobbs LV, Bandler RC, Streicher C, Cepko CL, Hippenmeyer S, Fishell G. Clonally Related Forebrain Interneurons Disperse Broadly across Both Functional Areas and Structural Boundaries. Neuron 2015;87(5):989-98.Abstract

The medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum, and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute.

Harwell CC, Fuentealba LC, Gonzalez-Cerrillo A, Parker PRL, Gertz CC, Mazzola E, Garcia MT, Alvarez-Buylla A, Cepko CL, Kriegstein AR. Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons. Neuron 2015;87(5):999-1007.Abstract

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Drokhlyansky E, Soh TK, Cepko CL. Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein. J Virol 2015;89(22):11718-22.Abstract

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.

Tang JCY, Rudolph S, Dhande OS, Abraira VE, Choi S, Lapan SW, Drew IR, Drokhlyansky E, Huberman AD, Regehr WG, Cepko CL. Cell type-specific manipulation with GFP-dependent Cre recombinase. Nat Neurosci 2015;18(9):1334-41.Abstract

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Zinn E, Pacouret S, Khaychuk V, Turunen HT, Carvalho LS, Andres-Mateos E, Shah S, Shelke R, Maurer AC, Plovie E, Xiao R, Vandenberghe LH. In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector. Cell Rep 2015;12(6):1056-68.Abstract

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Choi VW, Bigelow CE, McGee TL, Gujar AN, Li H, Hanks SM, Vrouvlianis J, Maker M, Leehy B, Zhang Y, Aranda J, Bounoutas G, Demirs JT, Yang J, Ornberg R, Wang Y, Martin W, Stout KR, Argentieri G, Grosenstein P, Diaz D, Turner O, Jaffee BD, Police SR, Dryja TP. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice. Mol Ther Methods Clin Dev 2015;2:15022.Abstract

Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year.

von Alpen D, Tran HV, Guex N, Venturini G, Munier FL, Schorderet DF, Haider NB, Escher P. Differential Dimerization of Variants Linked to Enhanced S-Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand-Binding Domain. Hum Mutat 2015;36(6):599-610.Abstract

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

Fuentealba LC, Rompani SB, Parraguez JI, Obernier K, Romero R, Cepko CL, Alvarez-Buylla A. Embryonic Origin of Postnatal Neural Stem Cells. Cell 2015;161(7):1644-55.Abstract

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

Conde P, Rodriguez M, van der Touw W, Jimenez A, Burns M, Miller J, Brahmachary M, Chen H-M, Boros P, Rausell-Palamos F, Yun TJ, Riquelme P, Rastrojo A, Aguado B, Stein-Streilein J, Tanaka M, Zhou L, Zhang J, Lowary TL, Ginhoux F, Park CG, Cheong C, Brody J, Turley SJ, Lira SA, Bronte V, Gordon S, Heeger PS, Merad M, Hutchinson J, Chen S-H, Ochando J. DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance. Immunity 2015;42(6):1143-58.Abstract

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Carvalho LS, Vandenberghe LH. Promising and delivering gene therapies for vision loss. Vision Res 2015;111(Pt B):124-33.Abstract
The maturity in our understanding of the genetics and the pathogenesis of disease in degenerative retinal disorders has intersected in past years with a novel treatment paradigm in which a genetic intervention may lead to sustained therapeutic benefit, and in some cases even restoration of vision. Here, we review this prospect of retinal gene therapy, discuss the enabling technologies that have led to first-in-human demonstrations of efficacy and safety, and the road that led to this exciting point in time.
Hou Y, Lin H, Zhu L, Liu Z, Hu F, Shi J, Yang T, Shi X, Guo H, Tan X, Zhang L, Wang Q, Li Z, Zhao Y. The inhibitory effect of IFN-γ on protease HTRA1 expression in rheumatoid arthritis. J Immunol 2014;193(1):130-8.Abstract
The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ-deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ-deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.

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