Biology subjects

Camarena, J.

Publications and source records attributed to Camarena, J..

8 recordsLinked to original sources

bioRxiv2024

Secreted Particle Information Transfer (SPIT) - A Cellular Platform For In Vivo Genetic Engineering

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cells nucleus, the ability of human cells to engraft into patients long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

synthetic biology↗

bioRxiv2023

Using human genetics to develop strategies to increase erythropoietic output from genome-edited hematopoietic stem and progenitor cells

Human genetic polymorphisms result in a diversity of phenotypes. Some sequences are pathologic and lead to monogenic diseases, while others may confer beneficial traits. Genome editing is a powerful tool to recreate genotypes found in the population, including the ability to correct pathologic mutations. One of the best characterized naturally occurring mutations causing congenital erythrocytosis arises from a truncation in the erythropoietin receptor (tEPOR) which can result in non-pathogenic hyper-production of red blood cells (RBCs). Using the precision of CRISPR/Cas9 genome editing, we have recreated tEPOR and studied the effect of variations of the genotype on RBC development. We then combined tEPOR with a correction strategy developed for {beta}-thalassemia and demonstrated that coupling the two genome editing events gave RBCs a significant selective advantage. This demonstrates the potential of combining human genetics with the precision of genome editing to enable safer and more effective genome editing therapies for patients with serious genetic diseases.

genetics↗

bioRxiv2021

Clinically Relevant Gene Editing in Hematopoietic Stem Cells for the Treatment of Pyruvate Kinase Deficiency Hemolytic Anemia

Pyruvate Kinase Deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene, which constitutes the main cause of chronic non-spherocytic hemolytic anemia. PKD incidence is estimated in 1 in 20,000 people worldwide. The PKLR gene encodes for the erythroid pyruvate kinase protein (RPK) implicated in the last step of the anaerobic glycolysis in red blood cells. The defective enzyme fails to maintain normal erythrocyte ATP levels, producing severe hemolytic anemia, and can be fatal in severe patients. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cells (HSPC) transplantation, so far. However, HSPC transplant is associated with a significant morbidity and mortality, especially in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR/Cas9 system and rAAVs for donor matrix delivery to build an efficient and safe system to knock-in a therapeutic donor at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient recipient mice. Moreover, erythroid cells derived from edited PKD-HSPCs restored normal levels of ATP, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene editing strategy may represent a lifelong therapy to restore RPK functionality in RBCs of patients and correct PKD. Single Sentence SummaryClinically relevant gene editing in hematopoietic stem cells for the treatment of Pyruvate Kinase Deficiency.

genetics↗

bioRxiv2020

Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells

{beta}-thalassemia pathology is not only due to loss of {beta}-globin (HBB), but also erythrotoxic accumulation and aggregation of the {beta}-globin binding partner, -globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in {beta}-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize {beta}-globin:-globin mRNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in mice, establishing proof-of-concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing {beta}-thalassemia.

molecular biology↗

bioRxiv2020

Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

developmental biology↗

bioRxiv2020

TRACE-Seq Reveals Clonal Reconstitution Dynamics of Gene Targeted Human Hematopoietic Stem Cells

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may usher in a new class of medicines to treat genetic diseases of the blood and immune system. With state-of-the-art methodologies, it is now possible to correct disease-causing mutations at high frequencies in HSPCs by combining ribonucleoprotein (RNP) delivery of Cas9 and chemically modified sgRNAs with homologous DNA donors via recombinant adeno-associated viral vector serotype six (AAV6). However, because of the precise nucleotide-resolution nature of gene correction, these current approaches do not allow for clonal tracking of gene targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a novel methodology that utilizes barcoded AAV6 donor template libraries, carrying either in-frame silent mutations or semi-randomized nucleotide sequences outside the coding region, to track the in vivo lineage contribution of gene targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of [~]20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in immunodeficient mice. We anticipate that this methodology has the potential to be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

molecular biology↗

bioRxiv2018

Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

Combination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

bioengineering↗

bioRxiv2018

Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureus homolog of Cas9) and SpCas9 (S. pyogenes homolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigen-specific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

immunology↗