Biology subjects

Lee, C. M.

Publications and source records attributed to Lee, C. M..

16 recordsLinked to original sources

bioRxiv2026

Dysregulation of epithelial differentiation architecture underlies field cancerization in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) has exceptionally high recurrence rates, exceeding 50%, contributing to mortality and morbidity. This recurrence rate is proposed to be due to field cancerization, a phenomenon where tumor-adjacent "field" tissue that appears histologically normal harbors molecular changes that prime it for subsequent tumor growth. Current models attribute field cancerization to shared driver mutations between tumors and adjacent at-risk tissue, but this assumption has never been directly tested. To comprehensively characterize this phenomenon, we profiled patient-matched tumor, normal-adjacent field (NAF), contralateral normal, and blood samples from HPV-negative HNSCC patients using whole genome sequencing, targeted driver gene sequencing, single-nucleus multiome profiling, spatial transcriptomics and functional proliferation assays. While histologically normal NAF tissues did harbor expanded mutant clones, these mutations rarely overlapped with paired tumors and were not shared across patients, suggesting molecular features beyond mutations are altered in NAF. Single-cell multiomics revealed that NAF epithelium loses its normal organizational hierarchy: basal stem cell markers persist in differentiated cells, while terminal differentiation programs fail to activate, with reconfiguration of both gene regulatory networks and cell-cell interactions. Proliferating cells appeared in tissue layers normally restricted to post-mitotic cells, a pattern typically seen only in overt dysplasia. Within this disrupted hierarchy, a distinct epithelial state arose in NAF that expressed both normal epithelial and tumor-associated markers, which we localized to the parabasal layers using immunofluorescence and spatial transcriptomics. Together, our findings demonstrate that field cancerization operates through epithelial hierarchy disorganization that disrupts differentiation, generates cells with tumor-like features, and may create a permissive environment for the expansion of cancer-associated mutations, all within tissue that appears histologically normal.

cancer biology↗

bioRxiv2026

NAE1-Dependent Protein Neddylation Preserves Endothelial Identity and Vascular Integrity

Background: Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis. Methods: We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury. Results: Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo. Conclusions: NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases.

pathology↗

bioRxiv2026

Circulating Immune Cells are Associated with Non-Inflammatory Pain in Rheumatoid Arthritis

Over half of patients with rheumatoid arthritis (RA) report clinically meaningful pain, despite treatment with disease-modifying antirheumatic drugs (DMARDs). While joint inflammation is a known cause of pain in patients with rheumatic diseases, emerging data indicate that many patients also suffer from centralized or nociplastic pain. There is a critical unmet need to characterize the altered cellular state that distinguishes patients with centralized pain. In the IMPACT study, 39 RA patients with minimal joint inflammation but varying levels of pain underwent quantitative sensory testing (QST) to assess nociplastic pain, completed patient-reported outcome (PRO) surveys, and provided blood samples for immune profiling. Supervised and unsupervised analysis of the multi-parameter spectral flow cytometry data identified immune cell populations correlated with nociplastic pain and patient-reported pain intensity. Moreover, analyses of single-cell RNA-seq from a subset of 22 patients revealed differences in cell type proportions and differential expression between the high and low pain groups. These studies provide novel insights into the role of circulating immune cells in altered central nervous system (CNS) pain regulation in adults with RA.

immunology↗

bioRxiv2026

Interrogating the Mechanisms of Cas9-mediated Allele Conversion

Allele conversion describes a process where a heterozygous variant is made homozygous. Recently, it has been shown that allele conversion can be triggered by DNA damage at the heterozygous site. This process has the potential to repair pathogenic heterozygous mutations; however, the efficiency is low. Here, we endeavoured to understand the mechanism underlying allele conversion, ultimately to raise allele conversion efficiency to functionally relevant levels. To test this, we developed a Compound Heterozygous Allele Conversion Reporter (CHACR) cell line. This line comprises knocked-in fluorescent protein encoding genes, with heterozygous inactivating mutations resulting in different fluorescence profiles from each allele. These mutations create protospacer adjacent motifs (PAM) for Cas9 recognition, where allele-specific gRNAs (AS-gRNAs) target the heterozygous mutations. We showed that applying these AS-gRNAs with either Cas9 nuclease or Cas9(D10A) nickase can recover mCherry fluorescence. Sorting and sequencing these fluorescent cells revealed wild-type sequences, suggesting allele conversion repaired the mutation using the homologous allele as a template. Allele conversion can also be triggered using an adenine base editor with an AS-gRNA, and this allele conversion mechanism can be manipulated by inhibiting DNA-PKcs or overexpressing RAD51. This work introduces a model for measuring allele conversion, and modifiers of this mechanism.

molecular biology↗

bioRxiv2025

Paleogenomic insight into the collapse, recovery, and management of American bison

American bison were pushed to the brink of extinction by the 20th century. This bottleneck and the fragmented nature of remnant populations pose challenges to their resilience, as does human-facilitated admixture between bison subspecies and with cattle. To contextualize current diversity, we sequenced 115 ancient and 45 modern bison genomes from across North America dating back within the last [~]20,000 years. Past bison populations were highly connected, in contrast to structured modern herds. Modern wood bison carry plains bison ancestry from 1920s translocations, while many bison lack cattle ancestry that has previously been believed to be ubiquitous. Our findings reveal the legacy of human impacts on bison in the context of modern conservation and highlight the applicability of ancient DNA for guiding wildlife restoration.

evolutionary biology↗

bioRxiv2025

Generation of Region-specific Airway Basal Stem Cells from Human Pluripotent Stem Cells via Regulation of BMP-NOGGIN Axis

Basal cells (BCs) are the primary stem cell population of adult human airways and a key target for hPSC-based models of airway development, disease and regenerative medicine. Recent studies have revealed substantial regional differences between human proximal and distal airway cell types, including BCs. Here, we show that the NOGGIN-BMP signaling axis governs proximal-distal patterning of hPSC-derived lung progenitors leading to the generation of region-specific induced BCs (iBCs). Continuous BMP inhibition through NOGGIN, coupled with tapered WNT activation, generates proximal iBCs that molecularly and functionally resemble human proximal airway BCs and differentiate in vitro and in vivo into the full repertoire of specialized proximal airway cell types, including ionocytes and pulmonary neuroendocrine cells. In sharp contrast, BMP activation with tapered WNT generates distal airway-like cells and distal BCs with limited ionocyte differentiation potential. Progeny of proximal iBCs derived from G551D CFTR mutant hiPSCs also recapitulate a cystic fibrosis-associated ionocyte phenotype.

cell biology↗

bioRxiv2025

Haplotype rather than single causal variants effects contribute to regulatory gene expression associations in human myeloid cells

Genome-wide association studies typically identify hundreds to thousands of loci, many of which harbor multiple independent peaks, each parsimoniously assumed to be due to the activity of a single causal variant. Fine-mapping of such variants has become a priority and since most associations are located within regulatory regions, it is also assumed that they colocalize with regulatory variants that influence the expression of nearby genes. Here we examine these assumptions by using a moderate throughput expression CROPseq protocol in which Cas9 nuclease is used to induce small insertions and deletions across the credible set of SNPs that may account for expression quantitative trait loci (eQTL) for genes associated with inflammatory bowel disease (IBD). Of the 4,384 SNPs targeted in 88 loci (an average of 50 per locus), 439 were significant and further examined for validation. From these, 98 significantly altered target gene expression in HL-60 myeloid cell line, 74 in induced macrophages from these HL-60 cells, and 78 in induced neutrophils for a total of 201 validated effects (46%), 43 of which were observed in at least two of the cell types. Considering the observed sensitivity and specificity of the controls, we estimate that there are at least 150 true positives per cell type, an average of almost 2.4 for each of the 64 eQTL for which putative causal variants have been fine-mapped. This implies that haplotype effects are likely to explain many of the associations. We also demonstrate that the same approach can be used to investigate the activity of very rare variants in regulatory regions for 89 genes, providing a rapid strategy for establishing clinical relevance of non-coding mutations.

genomics↗

bioRxiv2024

Sound-Evoked Plasticity Differentiates Tinnitus from Non-Tinnitus Mice

Tinnitus is the perception of non-meaningful sound in the absence of external stimuli. Although tinnitus behavior in animal models is associated with altered central nervous system activity, it is not currently possible to identify tinnitus using neuronal activity alone. In the mouse inferior colliculus (IC), a subpopulation of neurons demonstrates a sustained increase in spontaneous activity after a long-duration sound (LDS). Here, we use the "LDS test" to reveal tinnitus-specific differences in sound-evoked plasticity through IC extracellular recordings and the auditory brainstem response (ABRLDS) in CBA/CaJ mice after sound exposure and behavioral tinnitus assessment. Sound-exposed mice showed stronger and shorter tone-evoked responses in the IC compared to unexposed controls, but these differences were not strong predictors of tinnitus. In contrast, in the LDS test, non-tinnitus mice had a significantly stronger suppression in tone-evoked spike rate compared to tinnitus and unexposed control mice. ABR peak amplitudes also revealed robust differences between tinnitus and non-tinnitus mice, with ABR peaks from non-tinnitus mice exhibiting significantly stronger suppression in the LDS test compared to tinnitus and control mice. No significant differences were seen between cohorts in ABR amplitude, latency, wave V:I ratio, wave V:III ratio, I-V intra-peak latency, and I-VI intra-peak latency. We found high-frequency tone stimuli better suited to reveal tinnitus-specific differences for both extracellular IC and ABR recordings. We successfully used the LDS test to demonstrate that tinnitus-specific differences in sound-evoked plasticity can be shown using both multi-unit near-field recordings in the IC and non-invasive far-field recordings, providing a foundation for future electrophysiological research into the causes and treatment of tinnitus.

neuroscience↗

bioRxiv2024

A porcine chronic hepatitis E virus (HEV) infection model exhibits HEV replication in male accessory reproductive glands and immune-mediated reproductive damage

Hepatitis E virus (HEV) is an expanding zoonotic viral disease threat. Although HEV causes acute viral hepatitis, it is increasingly being recognized as a systemic pathogen with detection and damage in extrahepatic tissues. The presence of HEV RNA in the semen of chronically infected human patients in the absence of viremia and fecal shedding and presence of HEV in the sperm head underscores the need to understand the interaction of HEV within the male reproduction system. Male accessory glands secrete biofluids necessary for sperm nourishment and to neutralize the acidity of the vagina. The role of male accessory glands in the dissemination and persistence of HEV infection have not been studied. Using an immunosuppressed pig model for chronic HEV infection, we demonstrate infectious HEV in mature sperm cells altering the sperm motility and morphology. HEV isolated from sperm cells remained infectious in human hepatoma cells. Spermatic fluid contained lower virus titers than the sperm cells from chronically infected pigs highlighting that the sperm cells themselves can associate with the virus. Evaluation of the male accessory glands demonstrated viral replication, infiltration of CD45 leukocytes, and apoptosis associated with HEV infection. A decrease in serum testosterone levels was evident in the HEV infected pigs. Even though a lower viral RNA titer was seen in serum and feces of chronically infected, immunosuppressed and ribavirin treated pigs, high viral RNA and infectious particles in sperm is a concern. Our findings necessitate further studies defining the mechanism of sperm cell invasion by HEV, length of HEV survival in sperm cells during chronic HEV infection, and risk of sexual transmission of HEV during both acute and chronic phases of infection. Author SummaryHepatitis E virus, a leading cause of acute viral hepatitis, causes both acute and chronic infection in humans. Recent advances within the HEV field have demonstrated extrahepatic diseases associated with HEV. More recent findings have revealed infectious HEV in the vagina, Sertoli cells, and ejaculate of humans, and sperm cells of pigs. We demonstrate that the male accessory sex glands may have a role in the persistence of HEV infection during chronic infections. We utilized an established immunosuppressed pig model and treated pigs with ribavirin to study the presence of virus in the sperm cells. We demonstrated high viral RNA loads and infectious particles associated with sperm cells. Our study further highlights the importance of the testis, as an immune privileged site, in the maintenance of chronic HEV infection. New studies to evaluate the mechanisms by which HEV associates with sperm cells, the length of HEV survival in sperm cell fractions, and consideration of the testes as a potential HEV reservoir are necessary.

microbiology↗

bioRxiv2023

Rat hepatitis E virus (HEV) cross-species infection and transmission in pigs

Rocahepevirus ratti, an emerging hepatitis E virus (HEV), has recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs cohoused with naive pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to cohoused animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism. Significance StatementNew zoonotic strains of Rocahepevirus ratti (rat HEV) have emerged infecting both immunocompetent and immunosuppressed people through unknown transmission sources. Pigs are a primary source of transmission for human HEV strains and could be serving a similar role for rat HEV transmission as rats are a common pest found on swine farms worldwide. Rats could be transmitting rat HEV to pigs which could then be transmitted to humans. Determining susceptibility of pigs to emerging zoonotic rat HEV strains can define potentially new transmission routes to inform public health policy and could provide pathology models for rat HEV disease.

microbiology↗

bioRxiv2022

Mechanisms underlying the efficacy of a rodent model of vertical sleeve gastrectomy - a focus on energy expenditure

Background and aimsBariatric surgery remains the only effective and durable treatment option for morbid obesity. Vertical Sleeve Gastrectomy (VSG) is currently the most widely performed of these surgeries primarily because of its proven efficacy in generating rapid onset weight loss, improved glucose regulation and reduced mortality compared with other invasive procedures. VSG is associated with reduced appetite, however, the relative importance of energy expenditure to VSG-induced weight loss and changes in glucose regulation, particularly that in brown adipose tissue (BAT), remains unclear. The aim of this study is to investigate the role of BAT thermogenesis in the efficacy of VSG in a rodent model. MethodsDiet-induced obese male Sprague-Dawley rats were either sham-operated, underwent VSG surgery or were pairfed to the food consumed by the VSG group. Rats were also implanted with biotelemetry devices between the interscapular lobes of BAT to assess local changes in BAT temperature as a surrogate measure of thermogenic activity. Metabolic parameters including food intake, body weight and changes in body composition were assessed. To further elucidate the contribution of energy expenditure via BAT thermogenesis to VSG-induced weight loss, a separate cohort of lean rats underwent complete excision of the interscapular BAT (iBAT lipectomy) or chemical denervation using 6-hydroxydopamine (6-OHDA). To localize glucose uptake in specific tissues, an oral glucose tolerance test was combined with an intraperitoneal injection of 2 deoxy-D-glucose (2DG)-14C, administered intraperitoneally. Transneuronal viral tracing was used to identify 1) sensory neurons directed to the stomach or small intestine (H129-RFP) or 2) chains of polysynaptically linked neurons directed to BAT (PRV-GFP) in the same animals. ResultsFollowing VSG, there was a rapid reduction in body weight that was associated with reduced food intake, elevated BAT temperature and improved glucose regulation. Rats that underwent VSG had elevated glucose uptake into BAT compared to sham operated animals as well as elevated gene markers related to increased BAT activity (Ucp1, Dio2, Cpt1b, Cox8b, Ppargc) and markers of increased browning of white fat (Ucp1, Dio2, Cited1, Tbx1, Tnfrs9). Both iBAT lipectomy and 6-OHDA treatment significantly attenuated the impact of VSG on changes in body weight and adiposity in lean animals. In addition, surgical excision of iBAT following VSG significantly reversed VSG-mediated improvements in glucose tolerance, an effect that was independent of circulating insulin levels. Viral tracing studies highlight a patent neural link between the gut and BAT that include groups of premotor BAT-directed neurons in the dorsal raphe and raphe pallidus. ConclusionCollectively, these data support a role for BAT in mediating the metabolic sequelae, particularly the improvement in glucose regulation following VSG surgery and highlight the need to better understand the contribution from this tissue in human patients.

physiology↗

bioRxiv2022

Host Chitinase 3-like-1 is a Universal Therapeutic Target for the Delta, Omicron and Other SARS-CoV-2 Viral Variants in COVID 19

COVID 19 is the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; SC2) which has caused a world-wide pandemic with striking morbidity and mortality. Evaluation of SC2 strains demonstrated impressive genetic variability and many of these viral variants are now defined as variants of concern (VOC) that cause enhanced transmissibility, decreased susceptibility to antibody neutralization or therapeutics and or the ability to induce severe disease. Currently, the delta ({delta}) and omicron (o) variants are particularly problematic based on their impressive and unprecedented transmissibility and ability to cause break through infections. The delta variant also accumulates at high concentrations in host tissues and has caused waves of lethal disease. Because studies from our laboratory have demonstrated that chitinase 3-like-1 (CHI3L1) stimulates ACE2 and Spike (S) priming proteases that mediate SC2 infection, studies were undertaken to determine if interventions that target CHI3L1 are effective inhibitors of SC2 viral variant infection. Here we demonstrate that CHI3L1 augments epithelial cell infection by pseudoviruses that express the alpha, beta, gamma, delta or omicron S proteins and that the CHI3L1 inhibitors anti-CHI3L1 and kasugamycin inhibit epithelial cell infection by these VOC pseudovirus moieties. Thus, CHI3L1 is a universal, VOC-independent therapeutic target in COVID 19.

immunology↗

bioRxiv2021

Targeted replacement of full-length CFTR in human airway stem cells by CRISPR/Cas9 for pan-mutation correction in the endogenous locus

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

bioengineering↗

bioRxiv2020

Developmental PCB exposure disrupts synaptic transmission and connectivity in the rat auditory cortex, independent of its effects on peripheral hearing threshold

Polychlorinated biphenyls (PCBs) are enduring environmental toxicants and exposure is associated with neurodevelopmental deficits. The auditory system appears particularly sensitive, as previous work has shown that developmental PCB exposure causes both hearing loss and gross disruptions in the organization of the rat auditory cortex. However, the mechanisms underlying PCB-induced changes are not known, nor is it known if the central effects of PCBs are a consequence of peripheral hearing loss. Here, we study changes in both peripheral and central auditory function in rats with developmental PCB exposure using a combination of optical and electrophysiological approaches. Female rats were exposed to an environmental PCB mixture in utero and until weaning. At adulthood, auditory brainstem responses were measured, and synaptic currents were recorded in slices from auditory cortex layer 2/3 neurons. Spontaneous and miniature inhibitory postsynaptic currents (IPSCs) were more frequent in PCB-exposed rats compared to controls and the normal relationship between IPSC parameters and peripheral hearing was eliminated in PCB-exposed rats. No changes in spontaneous EPSCs were found. Conversely, when synaptic currents were evoked by laser photostimulation of caged-glutamate, PCB exposure did not affect evoked inhibitory transmission, but increased the total excitatory charge, the number and distance of sites that evoke a significant response. Together, these findings indicate that early developmental exposure to PCBs causes long-lasting changes in both inhibitory and excitatory neurotransmission in the auditory cortex that are independent of peripheral hearing changes, suggesting the effects are due to the direct impact of PCBs on the developing auditory cortex. Significance StatementThe mechanisms by which developmental exposure to polychlorinated biphenyls (PCBs) disrupt the central nervous system are not yet known. Here we show that developmental PCB exposure is associated with long-lasting dysregulation of both excitatory and inhibitory neurotransmission in the rodent brain. We further find that, unlike controls, synaptic parameters in the auditory cortex of PCB-exposed rats are independent of peripheral hearing changes. These data suggest that PCB-related changes in the auditory cortex are independent of their effects on the auditory periphery and that PCB exposure may disrupt the plastic mechanisms needed to restore normal processing in the auditory cortex after peripheral hearing loss.

neuroscience↗

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↗

bioRxiv2019

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the {Delta}F508 mutation (found in [~]70% of CF alleles and [~]90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous ({Delta}F508/{Delta}F508) and compound heterozygous ({Delta}F508/Other) CF patients, we achieved 28 {+/-} 5 % and 42 {+/-} 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41{+/-} 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31{+/-} 5 % in UABCs and 51 {+/-} 3 % in HBECs (both from subjects homozygous for {Delta}F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

bioengineering↗