Biology subjects

Likhite, S.

Publications and source records attributed to Likhite, S..

5 recordsLinked to original sources

bioRxiv2025

Gene replacement therapy for Piga GPI-anchor deficiency in the developing nervous system

Glycosylphosphatidylinositol (GPI) anchors are a class of post-translational modifications observed on over 150 proteins. Pathogenic variants in the GPI biosynthesis enzyme, PIGA, in humans are associated with several brain anomalies such as hypomyelination, cerebellar hypoplasia, ataxic gait, and can lead to premature mortality. We previously genetically deleted Piga from the embryonic mouse brain which led to early postnatal death and significant structural brain malformations similar to those observed in humans with PIGA variants. The current treatment options for PIGA patients only manage symptoms and provides palliative care, demonstrating a need for new therapeutic options. We employed an AAV9-mediated PIGA gene-replacement (AAV9-hPIGA) strategy to assess the efficacy of gene therapy in the brain. We show that a single intracerebroventricular treatment on the first day of life successfully rescued survival rates, structural brain anomalies, and neurological impairments. Additionally, we used mass spectrometry to identify and quantify GPI-anchored proteins in untreated and treated mutant mice. We found that AAV9- hPIGA treatment restored GPI-anchored protein levels in mutant animals. These investigations enhance our understanding of GPI-anchored protein production during brain development and contribute to the development of a more effective intervention for PIGA-related symptoms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/693709v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@32038dorg.highwire.dtl.DTLVardef@1875eb5org.highwire.dtl.DTLVardef@5abfe8org.highwire.dtl.DTLVardef@1ed5719_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIA single AAV9-hPIGA injection rescues Piga-related phenotypes including survival and structural brain defects C_LIO_LIAAV9-hPIGA treatment rescued long-term ataxia phenotypes in Piga mutants C_LIO_LINeuronal GPI-anchored protein levels in Piga mutants are increased with AAV9- hPIGA gene replacement C_LI

genetics↗

bioRxiv2021

AAV9-MCT8 delivery at juvenile stage ameliorates neurological and behavioral deficits in an AHDS mouse model

Allan-Herndon-Dudley syndrome (AHDS) is a severe X-linked intellectual and psychomotor disability disorder accompanied by abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in the monocarboxylate transporter 8 (MCT8), a specific TH transporter widely expressed in the central nervous system. MCT8 gene mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. AAV9-based gene therapy is a promising approach to treat monogenic neurological disorders. Here, the potential of this approach was tested in the well-established double knockout (dKO) Mct8-/y;Oatp1c1-/- mouse model of AHDS, which displays disease-relevant neurological and TH phenotypes. Systemic intravenous delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive function, as well as rescue of T3-brain content and T3-related gene expression. This preclinical study indicates that this gene therapy may improve the neurological symptoms of AHDS patients.

neuroscience↗

bioRxiv2021

CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3{zeta} and NKG2D/2B4+CD3{zeta}) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

immunology↗

bioRxiv2020

AAV9 gene therapy restores lifespan and treats pathological and behavioral abnormalities in a mouse model of CLN8-Batten disease

CLN8 disease is a rare form of neuronal ceroid lipofuscinosis caused by biallelic mutations in the CLN8 gene, which encodes a transmembrane endoplasmic reticulum protein involved in trafficking of lysosomal enzymes. CLN8 disease patients present with myoclonus, tonic-clonic seizures, and progressive declines in cognitive and motor function, with many cases resulting in premature death early in life. There are currently no treatments that can cure the disease or substantially slow disease progression. Using a mouse model of CLN8 disease, we tested the safety and efficacy of an intracerebroventricularly (ICV)-delivered self-complementary AAV9 (scAAV9) gene therapy vector driving expression of human CLN8. A single neonatal injection was safe and well-tolerated, resulting in robust transgene expression throughout the brain and spinal cord from 4 to 24 months, reducing histopathological and behavioral hallmarks of the disease and completely restoring lifespan from 10 months in untreated animals to beyond 24 months of age in treated animals. These results demonstrate, by far, the most successful rescue reported in an animal model of CLN8 disease, and supports gene therapy as a promising therapeutic strategy for this disorder.

neuroscience↗

bioRxiv2019

Highly efficient site-directed gene insertion in primary human natural killer cells using homologous recombination and CRISPaint delivered by AAV

Human peripheral blood natural killer (NK) cells have strong antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. However, genetic modification of NK cells has been challenging due to the high expression of innate sensing mechanisms for viral nucleic acids. Recently, we described an efficient vector-free method using Cas9/ribonucleoprotein complexes for gene deletion in NK cells. Here, we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a user-defined locus using homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. Using these approaches, we identified scAAV6 as the superior serotype for successful generation of stable mCherry-expressing primary NK cells (up to 89%). To maximize transgene packaging in HR-directed gene insertion, we identified minimum optimal homology arm lengths of 300bp for the flanking region of the Cas9-targeting site. Lastly, we demonstrate that mCherry positive NK cells can be expanded to large numbers using feeder cells expressing membrane-bound IL-21. This efficient method for site-directed insertion of genetic material into NK cells has broad potential for basic discovery and therapeutic applications for primary NK cells.

immunology↗