Biology subjects

Vonada, A.

Publications and source records attributed to Vonada, A..

3 recordsLinked to original sources

bioRxiv2025

Enhancement of Therapeutic Transgene Insertion for Murine Phenylketonuria

Low in vivo transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR)-mediated gene insertion as a treatment for Mendelian disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for the treatment of murine phenylalanine hydroxylase (PAH) deficiency, a model of human phenylketonuria (PKU), through pharmacologic inhibition of competing DNA repair pathways. Targeted integration of a Pah-expressing transgene into the hepatocytes of neonatal mice was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ). This was further improved following combination of vanillin and novobiocin, an inhibitor of microhomology-mediated end joining (MMEJ). Combined NHEJ and MMEJ inhibition yielded PAH-expressing transgene insertions in approximately 10% of targeted alleles and was associated with a 70.6% decrease in serum phenylalanine. Demonstrating that pharmacologic inhibition of DNA repair pathways that compete with HDR can significantly enhance HDR-mediated transgene insertion in vivo.

genomics↗

bioRxiv2023

Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation

Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria (PKU) by cell transplantation. Hepatocytes from a wildtype donor animal were edited in vitro to create Cypor deficiency and then transplanted into PKU animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from <1% to [~]14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for PKU with long-term efficacy and a favorable safety profile.

genetics↗

bioRxiv2022

Protein-coding evolution does not explain divergence in petal anthocyanin pigmentation between two monkeyflower species

Phenotypic transitions in related taxa often share a common genetic basis, which suggests that there are constraints that shape the process of evolution at the genetic level. For example, noncoding changes in a gene might be favored relative to coding changes due to being less constrained by pleiotropic effects. Here we evaluate the importance of coding-sequence changes to the recent evolution of a novel anthocyanin pigmentation trait in the monkeyflower genus Mimulus. The magenta-flowered Mimulus luteus var. variegatus recently gained petal lobe anthocyanin pigmentation via a single-locus Mendelian difference from its sister taxon, the yellow-flowered M. l. luteus. Previous work showed that the differentially expressed transcription factor gene MYB5a/NEGAN is the single causal gene. However, it was not clear whether MYB5a coding-sequence evolution (in addition to the observed patterns of differential expression) might also have contributed to increased anthocyanin production in M. l. variegatus. Quantitative image analysis of tobacco leaves, transfected with highly expressed MYB5a coding sequence from each taxon, revealed robust anthocyanin production driven by both alleles compared to a negative control. Counter to expectations, significantly higher anthocyanin production was driven by the coding sequence from the low-anthocyanin taxon M. l. luteus. Together with previously-published expression studies, this supports the hypothesis that petal pigment in M. l. variegatus was not gained by protein-coding changes, but instead via non- coding cis-regulatory evolution. Finally, while constructing the transgenes needed for this experiment, we unexpectedly discovered two sites in MYB5a that appear to be post- transcriptionally edited - a phenomenon that has been rarely reported, and even less often explored, for nuclear-encoded plant mRNAs.

molecular biology↗