Bio


I have extensive knowledge of genetics and have focused my research on inherited genetic mutations that act as disease-causing alleles for retinal degenerative disorders. I am skilled in modeling these complex human diseases using both genetically engineered mouse models as well as human cell lines, including embryonic and induced pluripotent stem cells. I have used these model systems I’ve developed to test therapies via microinjections into the mouse (both in utero during embryogenesis and via the subretinal/intravitreal space in the eye). I initially focused my therapeutic studies on gene correction, both by gene editing (CRISPR/Cas9, TALEN, or ZFN) into the human cells or mouse blastocysts to correct genetic mutations or providing therapeutic agents as viruses (I have used both lentivirus and AAV). Recently, I am interested in utilizing my knowledge of nutrition and metabolic biology for metabolic rescue as a therapeutic for patients with inherited vitreoretinopathies regardless of their genetic mutations. Additionally, I have transplanted gene edited human cells into the mouse eye to test for both the functional ability of the human cells in a live context, and as a method of pre-clinical studies for therapeutic efficacy of human cell transplantation. My overall research goal is to discover and understand the genetic mechanisms underlying neuro-retinal disease, and to provide novel therapeutics for these complex degenerative disorders using gene therapy and genome engineering technologies, human stem cell transplantations, and metabolic rescue.

Honors & Awards


  • Invited Speaker, 60th Anniversary Symposium, Institute of Human Nutrition, Columbia University (2017)
  • The Takashi Murachi Award for the outstanding presentation by a post-doctoral trainee, Federation of American Societies for Experimental Biology (FASEB) (2016)
  • Session Co-Chair, the Biology of Calpains in Health and Disease Science Research Conference, Federation of American Societies for Experimental Biology (FASEB) (2016)
  • Invited Speaker, the Biology of Calpains in Health and Disease Science Research Conference, Federation of American Societies for Experimental Biology (FASEB) (2016)
  • Director’s Fellowship Allowance, The Whitehead Institute for Biomedical Research (2015)
  • The Bernard F. Erlanger Award for Excellence in Research, Columbia University (2014)
  • Pass with Honors, Thesis Dissertation and Defense, Columbia University (2013)
  • Fellow Teaching Credential, New York Academy of Sciences (2013)
  • Travel Grant for the Annual Association for Research in Vision and Ophthalmology (ARVO) Conference, Columbia University Graduate Student Advisory Council (2013)
  • Marija Dokmanovic Chouinard Award for Excellence in Research, Columbia University (2012)
  • Charles J. Epstein Trainee Award for Excellence in Human Genetics Research – Finalist, American Society of Human Genetics (ASHG) (2012)
  • B. Dobli Srinivasan, M.D. Award, Best Resident/Fellow Basic Science Research Paper, Columbia University (2012)
  • Scholarship toward the Medical and Experimental Mammalian Genetics Course at Jax, March of Dimes (2012)
  • Travel Grant for the Mouse Development, Genetics and Genomics Meeting at CSHL, Burroughs-Wellcome Fund (2010)
  • Fire Department of New York (FDNY) C-14 Certificate of Fitness in Lab Safety, Columbia University (2010)
  • B. Dobli Srinivasan, M.D. Award, Best Resident/Fellow Clinical Research Paper, Columbia University (2009)
  • Graduated Cum Laude, Covenant College (2008)
  • Annual Dean’s List, Covenant College (2004-2008)

Boards, Advisory Committees, Professional Organizations


  • Community Leader for the Junior Academy, New York Academy of Sciences Global STEM Alliance (2017 - Present)
  • Mentor, Next Scholars Program, New York Academy of Sciences Global STEM Alliance (2017 - Present)
  • Junior Academy Master Mentor, Mentee Candidate Application Reviewer, Mentor Candidate Interviewer, and Independent Consultant, New York Academy of Sciences Global STEM Alliance (2016 - Present)
  • Mentor, 1000 Girls, 1000 Futures Program, New York Academy Global STEM Alliance (2016 - Present)
  • Mentor, Junior Academy, New York Academy of Sciences Global STEM Alliance (2016 - Present)
  • Mentor, After-school STEM Mentoring Program, Genetics Laboratory, 4th-7th Grade Girl Scout Juniors/Cadettes, New York, NY, New York Academy of Sciences (2013 - 2013)
  • Mentor, After-school STEM Mentoring Program, Genetics Laboratory, 4th Grade Children at the Hudson Guild Community Center, New York, NY, New York Academy of Sciences (2012 - 2012)

Professional Education


  • Post-doctoral Fellow, The Whitehead Institute for Biomedical Research/MIT, Stem Cell and Developmental Biology (2017)
  • Doctor of Philosophy, Columbia University (2013)
  • Master of Philosophy, Columbia University (2011)
  • Master of Science, Columbia University (2009)
  • Bachelor of Arts, Covenant College (2008)

Lab Affiliations


All Publications


  • In Vivo Expression of Mutant Calpains in the Eye Using Lentivirus. Methods in molecular biology (Clifton, N.J.) Wert, K. J., Mahajan, V. B. 2019; 1915: 233–47

    Abstract

    Exome sequencing has identified many candidate genes and mutations for human diseases, but the functional validation of these candidates is a time-consuming and costly process. Here, we describe a method which uses lentiviruses to overexpress calpain mutations that may play a role in dominant diseases such as autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). The use of lentivirus to deliver the mutant calpain allows for a cost-effective, rapid, and efficient approach to test whether or not a candidate gene mutation from exome sequencing acts as the disease-causing allele for a human disorder. This method also provides for a comparison of different candidate mutations from a single gene identified by exome sequencing, as well as elucidating the mechanisms underlying these complex human disorders. Furthermore, this chapter focuses on two different methods to deliver mutant calpain to the cells of the eye, using either a subretinal or an intravitreal injection of the lentivirus into the mouse eye.

    View details for PubMedID 30617808

  • Insights into Retinal Development Using Live Imaging in Female Carriers of Choroideremia. Ophthalmic surgery, lasers & imaging retina Wert, K. J., Bakall, B., Bassuk, A. G., Tsang, S. H., Mahajan, V. B. 2019; 50 (5): e158–e162

    Abstract

    Lineage tracing can provide key insights into the development of tissues, such as the retina. Yet it is not possible to manipulate human cells during embryogenesis. The authors observed a distinct phenotype in female carriers of X-linked disorders, in particular, carriers of choroideremia caused by mutations in CHM, encoding Rab escort protein-1. The authors found that X chromosome inactivation provides a method for retinal lineage tracing in human patients. Live imaging of female carriers displays a developmental pattern that is different within the peripheral retina compared with the posterior retina and provides important insights into the development and migration of retinal cells. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:e158-e162.].

    View details for DOI 10.3928/23258160-20190503-15

    View details for PubMedID 31100169

  • Extracellular superoxide dismutase 3 (SOD3) regulates oxidative stress at the vitreoretinal interface. Free radical biology & medicine Wert, K. J., Velez, G., Cross, M. R., Wagner, B. A., Teoh-Fitzgerald, M. L., Buettner, G. R., McAnany, J., Olivier, A., Tsang, S. H., Harper, M. M., Domann, F. E., Bassuk, A. G., Mahajan, V. B. 2018

    Abstract

    Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases.

    View details for PubMedID 29940351

  • Establishment of human pluripotent stem cell-derived pancreatic β-like cells in the mouse pancreas. Proceedings of the National Academy of Sciences of the United States of America Ma, H., Wert, K. J., Shvartsman, D., Melton, D. A., Jaenisch, R. 2018

    Abstract

    Type 1 diabetes is characterized by autoimmune destruction of β cells located in pancreatic islets. However, tractable in vivo models of human pancreatic β cells have been limited. Here, we generated xenogeneic human pancreatic β-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived β (SC-β) cells into the pancreas of neonatal mice. The engrafted β-like cells expressed β cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to β-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-β cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic β-like cells in the endogenous niche.

    View details for DOI 10.1073/pnas.1702059115

    View details for PubMedID 29599125

  • Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis. PloS one Bakthavatchalu, V., Wert, K. J., Feng, Y., Mannion, A., Ge, Z., Garcia, A., Scott, K. E., Caron, T. J., Madden, C. M., Jacobsen, J. T., Victora, G., Jaenisch, R., Fox, J. G. 2018; 13 (3): e0194443

    Abstract

    Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

    View details for DOI 10.1371/journal.pone.0194443

    View details for PubMedID 29554148

    View details for PubMedCentralID PMC5858775

  • Molecular Criteria for Defining the Naive Human Pluripotent State CELL STEM CELL Theunissen, T. W., Friedli, M., He, Y., Planet, E., O'Neil, R. C., Markoulaki, S., Pontis, J., Wang, H., Iouranova, A., Imbeault, M., Duc, J., Cohen, M. A., Wert, K. J., Castanon, R., Zhang, Z., Huang, Y., Nery, J. R., Drotar, J., Lungjangwa, T., Trono, D., Ecker, J. R., Jaenisch, R. 2016; 19 (4): 502-515

    Abstract

    Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.

    View details for DOI 10.1016/j.stem.2016.06.011

    View details for Web of Science ID 000389473800013

    View details for PubMedID 27424783

    View details for PubMedCentralID PMC5065525

  • Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, M. A., Wert, K. J., Goldmann, J., Markoulaki, S., Buganim, Y., Fu, D., Jaenisch, R. 2016; 113 (6): 1570-1575

    Abstract

    The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W(sh)/W(sh)), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases.

    View details for DOI 10.1073/pnas.1525518113

    View details for Web of Science ID 000369571700043

    View details for PubMedID 26811475

    View details for PubMedCentralID PMC4760776

  • Pathology and mechanism of eye diseases. Ophthalmic Disease Mechanisms and Drug Discovery Wert, K. J., Kroeger, H., Wu, F., Tsang, S. H., Lin, J. H. 2016: 3–32
  • Neuroretinal hypoxic signaling in a new preclinical murine model for proliferative diabetic retinopathy SIGNAL TRANSDUCTION AND TARGETED THERAPY Wert, K. J., Mahajan, V. B., Zhang, L., Yan, Y., Li, Y., Tosi, J., Hsu, C., Nagasaki, T., Janisch, K. M., Grant, M. B., Mahajan, M., Bassuk, A. G., Tsang, S. H. 2016; 1
  • Neuroretinal hypoxic signaling in a new preclinical murine model for proliferative diabetic retinopathy. Signal transduction and targeted therapy Wert, K. J., Mahajan, V. B., Zhang, L., Yan, Y., Li, Y., Tosi, J., Hsu, C. W., Nagasaki, T., Janisch, K. M., Grant, M. B., Mahajan, M., Bassuk, A. G., Tsang, S. H. 2016; 1

    Abstract

    Diabetic retinopathy (DR) affects approximately one-third of diabetic patients and, if left untreated, progresses to proliferative DR (PDR) with associated vitreous hemorrhage, retinal detachment, iris neovascularization, glaucoma and irreversible blindness. In vitreous samples of human patients with PDR, we found elevated levels of hypoxia inducible factor 1 alpha (HIF1α). HIFs are transcription factors that promote hypoxia adaptation and have important functional roles in a wide range of ischemic and inflammatory diseases. To recreate the human PDR phenotype for a preclinical animal model, we generated a mouse with neuroretinal-specific loss of the von Hippel Lindau tumor suppressor protein, a protein that targets HIF1α for ubiquitination. We found that the neuroretinal cells in these mice overexpressed HIF1α and developed severe, irreversible ischemic retinopathy that has features of human PDR. Rapid progression of retinopathy in these mutant mice should facilitate the evaluation of therapeutic agents for ischemic and inflammatory blinding disorders. In addition, this model system can be used to manipulate the modulation of the hypoxia signaling pathways, for the treatment of non-ocular ischemic and inflammatory disorders.

    View details for PubMedID 27195131

  • A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity STEM CELL REPORTS D'Alessio, A. C., Fan, Z. P., Wert, K. J., Baranov, P., Cohen, M. A., Saini, J. S., Cohick, E., Charniga, C., Dadon, D., Hannett, N. M., Young, M. J., Temple, S., Jaenisch, R., Lee, T. I., Young, R. A. 2015; 5 (5): 763-775

    Abstract

    Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.

    View details for DOI 10.1016/j.stemcr.2015.09.016

    View details for Web of Science ID 000364991000009

    View details for PubMedID 26603904

    View details for PubMedCentralID PMC4649293

  • CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model HUMAN MOLECULAR GENETICS Wert, K. J., Bassuk, A. G., Wu, W., Gakhar, L., Coglan, D., Mahajan, M., Wu, S., Yang, J., Lin, C., Tsang, S. H., Mahajan, V. B. 2015; 24 (16): 4584-4598

    Abstract

    A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.

    View details for DOI 10.1093/hmg/ddv189

    View details for Web of Science ID 000361315400009

    View details for PubMedID 25994508

    View details for PubMedCentralID PMC4512628

  • Silencing of tuberin enhances photoreceptor survival and function in a preclinical model of retinitis pigmentosa (an american ophthalmological society thesis). Transactions of the American Ophthalmological Society Tsang, S. H., Chan, L., Tsai, Y., Wu, W., Hsu, C., Yang, J., Tosi, J., Wert, K. J., Davis, R. J., Mahajan, V. B. 2014; 112: 103-115

    Abstract

    To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis.In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin's role in regulating survival.At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration.Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.

    View details for PubMedID 25646031

  • Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina HUMAN MOLECULAR GENETICS Wert, K. J., Skeie, J. M., Bassuk, A. G., Olivier, A. K., Tsang, S. H., Mahajan, V. B. 2014; 23 (10): 2665-2677

    Abstract

    Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing.

    View details for DOI 10.1093/hmg/ddt661

    View details for Web of Science ID 000334694800013

    View details for PubMedID 24381307

    View details for PubMedCentralID PMC3990166

  • Mid-stage intervention achieves similar efficacy as conventional early-stage treatment using gene therapy in a pre-clinical model of retinitis pigmentosa HUMAN MOLECULAR GENETICS Wert, K. J., Sancho-Pelluz, J., Tsang, S. H. 2014; 23 (2): 514-523

    Abstract

    Deficiencies in rod-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase-6 (PDE6) are the third most common cause of autosomal recessive retinitis pigmentosa (RP). Previously, viral gene therapy approaches on pre-clinical models with mutations in PDE6 have demonstrated that the photoreceptor cell survival and visual function can be rescued when the gene therapy virus is delivered into the subretinal space before the onset of disease. However, no studies have currently been published that analyze rescue effects after disease onset, a time when human RP patients are diagnosed by a clinician and would receive the treatment. We utilized the AAV2/8(Y733F)-Rho-Pde6α gene therapy virus and injected it into a pre-clinical model of RP with a mutation within the alpha subunit of PDE6: Pde6α(D670G). These mice were previously shown to have long-term photoreceptor cell rescue when this gene therapy virus was delivered before the onset of disease. Now, we have determined that subretinal transduction of this rod-specific transgene at post-natal day (P) 21, when approximately half of the photoreceptor cells have undergone degeneration, is more efficient in rescuing cone than rod photoreceptor function long term. Therefore, AAV2/8(Y733F)-Rho-Pde6α is an effective gene therapy treatment that can be utilized in the clinical setting, in human patients who have lost portions of their peripheral visual field and are in the mid-stage of disease when they first present to an eye-care professional.

    View details for DOI 10.1093/hmg/ddt452

    View details for Web of Science ID 000330840400019

    View details for PubMedID 24101599

    View details for PubMedCentralID PMC3869365

  • General pathophysiology in retinal degeneration. Developments in ophthalmology Wert, K. J., Lin, J. H., Tsang, S. H. 2014; 53: 33-43

    Abstract

    Retinal degeneration, including that seen in age-related macular degeneration and retinitis pigmentosa (RP), is the most common form of neural degenerative disease in the world. There is great genetic and allelic heterogeneity of the various retinal dystrophies. Classifications of these diseases can be ambiguous, as there are similar clinical presentations in retinal degenerations arising from different genetic mechanisms. As would be expected, alterations in the activity of the phototransduction cascade, such as changes affecting the renewal and shedding of the photoreceptor OS, visual transduction, and/or retinol metabolism have a great impact on the health of the retina. Mutations within any of the molecules responsible for these visual processes cause several types of retinal and retinal pigment epithelium degenerative diseases. Apoptosis has been implicated in the rod cell loss seen in a mouse model of RP, but the precise mechanisms that connect the activation of these pathways to the loss of phosphodiesterase (PDE6β) function has yet to be defined. Additionally, the activation of apoptosis by CCAAT/-enhancer-binding protein homologous protein (CHOP), after activation of the unfolded protein response pathway, may be responsible for cell death, although the mechanism remains unknown. However, the mechanisms of cell death after loss of function of PDE6, which is a commonly studied mammalian model in research, may be generalizable to loss of function of different key proteins involved in the phototransduction cascade.

    View details for DOI 10.1159/000357294

    View details for PubMedID 24732759

    View details for PubMedCentralID PMC4405532

  • Therapeutic Margins in a Novel Preclinical Model of Retinitis Pigmentosa JOURNAL OF NEUROSCIENCE Davis, R. J., Hsu, C., Tsai, Y., Wert, K. J., Sancho-Pelluz, J., Lin, C., Tsang, S. H. 2013; 33 (33): 13475-13483

    Abstract

    The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, Pde6b(Stop), which allows us to temporally correct PDE6-deficiency. Whereas untreated mutants exhibit degeneration, activation of Cre-loxP recombination in early embryogenesis produced stable long-term rescue. Reversal at later time-points showed partial long-term or short-lived rescue. Our results suggest stable restoration of retinal function by gene therapy can be achieved if a sufficient number of rods are treated. Because patients are generally diagnosed after extensive loss of rods, the success of clinical trials may depend on identifying patients as early as possible to maximize the number of treatable rods.

    View details for DOI 10.1523/JNEUROSCI.0419-13.2013

    View details for Web of Science ID 000323155700022

    View details for PubMedID 23946405

    View details for PubMedCentralID PMC3742933

  • Functional rescue for a full year after gene therapy in a pre-clinical model of retinitis pigmentosa Wert, K., Davis, R., Sancho-Pelluz, J., Lin, C., Tsang, S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2013
  • Translational Modeling of Calpain-5 Vitreoretinopathy Mechanisms in Mice Mahajan, V., Wert, K., Skeie, J., Tsang, S. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2013
  • Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa HUMAN MOLECULAR GENETICS Wert, K. J., Davis, R. J., Sancho-Pelluz, J., Nishina, P. M., Tsang, S. H. 2013; 22 (3): 558-567

    Abstract

    Approximately 36 000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are caused by a loss in phosphodiesterase (PDE6) function. In the preclinical Pde6α(nmf363) mouse model of this disease, defects in the α-subunit of PDE6 result in a progressive loss of photoreceptors and neuronal function. We hypothesized that increasing PDE6α levels using an AAV2/8 gene therapy vector could improve photoreceptor survival and retinal function. We utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)-Rho-Pde6α, to transduce Pde6α(nmf363) retinas and monitored its effects over a 6-month period (a quarter of the mouse lifespan). We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. These results suggest that RP due to PDE6α deficiency in humans, in addition to PDE6β deficiency, is also likely to be treatable by gene therapy.

    View details for DOI 10.1093/hmg/dds466

    View details for Web of Science ID 000313531500012

    View details for PubMedID 23108158

    View details for PubMedCentralID PMC3542865

  • Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye. Journal of visualized experiments : JoVE Wert, K. J., Skeie, J. M., Davis, R. J., Tsang, S. H., Mahajan, V. B. 2012

    Abstract

    The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.(1) Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.(2) These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.(3-4) Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.(5-8) In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

    View details for DOI 10.3791/4286

    View details for PubMedID 23207897

  • Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Wert, K. J., Skeie, J. M., Davis, R. J., Tsang, S. H., Mahajan, V. B. 2012

    Abstract

    The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.(1) Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.(2) These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.(3-4) Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.(5-8) In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

    View details for DOI 10.3791/4286

    View details for Web of Science ID 000209226000027

    View details for PubMedCentralID PMC3578262

  • DEOXYGENATION OF AROMATIC KETONES USING TRANSFER HYDROGENOLYSIS WITH RANEY NICKEL IN 2-PROPANOL SYNTHETIC COMMUNICATIONS Zuidema, D. R., Williams, S. L., Wert, K. J., Bosma, K. J., Smith, A. L., Mebane, R. C. 2011; 41 (19): 2927-2931
  • Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for Retinitis Pigmentosa: Transplantation 2010 April 27;89 (8): 911-919. Annals of neurosciences Nan-Kai, W., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R., Lai, C., Chien, C., Nagasaki, T., Lin, C., Tsang, S. H. 2010; 17 (4): 185-186

    View details for DOI 10.5214/ans.0972.7531.1017408

    View details for PubMedID 25205903

    View details for PubMedCentralID PMC4117017

  • Transplantation of Reprogrammed Embryonic Stem Cells Improves Visual Function in a Mouse Model for Retinitis Pigmentosa TRANSPLANTATION Wang, N., Tosi, J., Kasanuki, J. M., Chou, C. L., Kong, J., Parmalee, N., Wert, K. J., Allikmets, R., Lai, C., Chien, C., Nagasaki, T., Lin, C., Tsang, S. H. 2010; 89 (8): 911-919

    Abstract

    To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice.Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation.RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue.ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

    View details for DOI 10.1097/TP.0b013e3181d45a61

    View details for Web of Science ID 000277220200002

    View details for PubMedID 20164818

    View details for PubMedCentralID PMC2855750

  • Novel Method of Reducing Ketones Using Sodium Hydroxide in Isopropanol SYNTHETIC COMMUNICATIONS Zuidema, D. R., Wert, K. J., Williams, S. L., Chill, S. T., Holte, K. L., Kokes, N. K., Mebane, R. C. 2010; 40 (8): 1187-1191