Biology subjects

Glazer, P. M.

Publications and source records attributed to Glazer, P. M..

4 recordsLinked to original sources

bioRxiv2024

Systemic in utero gene editing as a treatment for cystic fibrosis

In utero gene editing has the potential to modify disease causing genes in multiple developing tissues before birth, possibly allowing for normal organ development, disease improvement, and conceivably, cure. In cystic fibrosis (CF), a disease that arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, there are signs of multiorgan disease affecting the function of the respiratory, gastrointestinal, and reproductive systems already present at birth. Thus, treating CF patients early is crucial for preventing or delaying irreversible organ damage. Here we demonstrate proof-of-concept of multiorgan mutation correction in CF using peptide nucleic acids (PNAs) encapsulated in polymeric nanoparticles and delivered systemically in utero. In utero editing was associated with sustained postnatal CFTR activity, at a level similar to that of wild-type mice, in both respiratory and gastrointestinal tissue, without detection of off-target mutations in partially homologous loci. This work suggests that systemic in utero gene editing represents a viable strategy for treating monogenic diseases before birth that impact multiple tissue types.

bioengineering↗

bioRxiv2024

Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

Radiotherapy (RT), is a fundamental treatment for malignant tumors and is used in over half of cancer patients. As radiation can promote anti-tumor immune effects, a promising therapeutic strategy is to combine radiation with immune checkpoint inhibitors (ICIs). However, the genetic determinants that impact therapeutic response in the context of combination therapy with radiation and ICI have not been systematically investigated. To unbiasedly identify the tumor intrinsic genetic factors governing such responses, we performed a set of genome-scale CRISPR screens in melanoma cells for cancer survival in response to low-dose genotoxic radiation treatment, in the context of CD8 T cell co-culture and with anti-PD1 checkpoint blockade antibody. Two actin capping proteins, Capza3 and Capg, emerged as top hits that upon inactivation promote the survival of melanoma cells in such settings. Capza3 and Capg knockouts (KOs) in mouse and human cancer cells display persistent DNA damage due to impaired homology directed repair (HDR); along with increased radiation, chemotherapy, and DNA repair inhibitor sensitivity. However, when cancer cells with these genes inactivated were exposed to sublethal radiation, the persistence of DNA damage promoted activation of the STING pathway, induction of inhibitory CEACAM1 ligand expression and resistance to CD8 T cell killing. Patient cancer genomics analysis reveals an increased mutational burden in patients with inactivating mutations in CAPG and/or CAPZA3, at levels comparable to other HDR associated genes. There is also a positive correlation between CAPG expression, CD8 T cell tumor infiltration and response to immunotherapy among patient datasets. Our results unveil the critical roles of actin binding proteins for efficient HDR within cancer cells and demonstrate a previously unrecognized regulatory mechanism of therapeutic response to radiation and immunotherapy.

genetics↗

bioRxiv2023

DNA Recognition and Induced Genome Modification by a Hydroxymethyl-γ Tail-Clamp Peptide Nucleic Acid

Peptide nucleic acids (PNA) can target and stimulate recombination reactions in genomic DNA. We have reported that gamma ({gamma})-PNA oligomers possessing the diethylene glycol {gamma}-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve {gamma}PNA accessibility, we explore the utility of {gamma}PNA oligomers possessing the hydroxymethyl-{gamma} moiety for gene editing applications. We demonstrate that a {gamma}PNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity to that of the diethylene glycol-{gamma}PNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-{gamma}PNA stimulates higher frequencies ([≥] 1.5-fold) of gene modification than the diethylene glycol {gamma}PNA in mouse bone marrow cells.

genetics↗

bioRxiv2018

Pathological Oxidation of PTPN12 Underlies ABL1 Phosphorylation in HLRCC

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germ line inactivating alterations in Fumarate Hydratase (FH) cause HLRCC, and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH -/-PRCC cells have increased ABL1 activation, which promotes tumor growth, but how ABL1 is activated remained unclear. Oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity; conceivably, ROS-catalyzed inactivation of an ABL-directed PTP might account for ABL1 activation in this malignancy. Previously, our group developed \"q-oxPTPome,\" a method that can globally monitor the oxidation of classical PTPs. We have now refined the q-oxPTPome approach, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models shows that multiple PTPs are either highly oxidized (including PTPN12) or overexpressed. In general, highly oxidized PTPs were those that have relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells is reversible, although nearly 40% of PTPN13 is oxidized irreversibly to the sulfonic acid state. Using \"substrate-trapping mutants\", we mapped PTPs to their putative substrates, and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identify PTPN12 as the major ABL1 phosphatase in HLRCC. Overall, our results show that ROS-induced PTPN12 oxidation accounts for ABL1 phosphorylation in HLRCC-associated PRCC, reveal a novel mechanism for inactivating a tumor suppressor gene product, and establish a direct link between pathological PTP oxidation and neoplastic disease.

cancer biology↗