Disruption of Endosomal Membrane by Cationic Vectors Drives Endosomal Release and Enables Successful Gene Delivery

The potential of gene therapy to treat congenital disorders is hindered by the lack of safe and effective delivery agents (vectors). More effective viral vectors possess several safety concerns but the safer non-viral vectors are less effective in inducing gene expression. Understanding how vector-DNA complexes (polyplexes) are internalized and transported to the nucleus would help make effective non-viral vectors. Several processes including 1) endosomal release, 2) transport within specific intracellular pathways, 3) protection of DNA from nucleases and 4) transport into the nucleus have been identified to affect gene delivery. However, the relative importance of these processes and their relationship to vector properties are unknown. Our experiments indicate that the endosomal release of polyplexes, due to the disruption of endosomal membranes mediated by the intercalation of free cationic vectors, is critical for successful gene delivery. This model was developed using two studies that 1) tracked the transport of intact DNA in cells and 2) quantified vector-cell membrane interactions. Specifically, present imaging techniques cannot distinguish functional intact DNA from the vast majority of degraded DNA. To distinguish intact DNA from degraded DNA, we used a novel DNA oligo nucleotide molecular beacon (OMB) labeled with a dye pair that exhibits Forester Resonance Energy Transfer (FRET). We observed that the fraction of cells displaying release of intact DNA from endosomes quantitatively predicted the fraction of cells displaying gene expression for effective (jetPEI) and ineffective cationic vectors (G5-PAMAM). Moreover, intact OMB delivered with G5-PAMAM and confined to endosomes could be released by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. Thus, microscopy studies indicated that free cationic vectors drive endosomal release. Moreover, experiments quantifying vector/cell membrane interactions further showed that the ability of free vectors , not polyplexes, to permeabilize cell membranes predicted successful gene expression. In summary, our experiments provide a novel new strategy that emphasizes the role of cell membrane-cationic vector interactions in driving successful gene expression.PhDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120751/1/svaidy_1.pd

Cyclic orthogonal double covers of 6-regular circulant graphs by disconnected forests

An orthogonal double cover (ODC) of a graph H is a collection G = {Gv : v ∈ V (H)} of |V (H)| subgraphs of H such that every edge of H is contained in exactly two members of G and for any two members Gu and Gv in G, |E(Gu) ∩ E(Gv)| is 1 if u and v are adjacent in H and it is 0 if u and v are nonadjacent in H. An ODC G of H is cyclic if the cyclic group of order |V (H)| is a subgroup of the automorphism group of G; otherwise it is noncyclic. Recently, Sampathkumar and Srinivasan settled the problem of the existence of cyclic ODCs of 4-regular circulant graphs. An ODC G of H is cyclic (CODC) if the cyclic group of order | V (H)| is a subgroup of the automorphism group of G, the set of all automorphisms of G; otherwise it is noncyclic. In this paper, we have completely settled the existence problem of CODCs of 6-regular circulant graphs by four acyclic disconnected graphs.Publisher's Versio

Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses

Second-look PET-CT following an initial incomplete PET-CT response to (chemo)radiotherapy for head and neck squamous cell carcinoma

OBJECTIVES: The limited positive predictive value of an incomplete response on PET-CT following (chemo)radiotherapy for head and neck squamous cell carcinoma (HNSCC) means that the optimal management strategy remains uncertain. The aim of the study is to assess the utility of a 'second-look' interval PET-CT. METHODS: Patients with HNSCC who were treated with (chemo)radiotherapy between 2008 and 2017 and underwent (i) baseline and (ii) response assessment PET-CT and (iii) second-look PET-CT following incomplete (positive or equivocal scan) response were included. Endpoints were conversion rate to complete response (CR) and test characteristics of the second-look PET-CT. RESULTS: Five hundred sixty-two patients with HNSCC underwent response assessment PET-CT at a median of 17 weeks post-radiotherapy. Following an incomplete response on PET-CT, 40 patients underwent a second-look PET-CT at a median of 13 weeks (range 6-25) from the first response PET-CT. Thirty-four out of 40 (85%) patients had oropharyngeal carcinoma. Twenty-four out of 40 (60%) second-look PET-CT scans converted to a complete locoregional response. The primary tumour conversion rate was 15/27 (56%) and the lymph node conversion rate was 14/19 (74%). The sensitivity, specificity, positive predictive value and negative predictive value (NPV) of the second-look PET-CT were 75%, 75%, 25% and 96% for the primary tumour and 100%, 92%, 40% and 100% for lymph nodes. There were no cases of progression following conversion to CR in the primary site or lymph nodes. CONCLUSIONS: The majority of patients who undergo a second-look PET-CT convert to a CR. The NPV of a second-look PET-CT is high, suggesting the potential to avoid surgical intervention. KEY POINTS: • PET-CT is a useful tool for response assessment following (chemo)radiotherapy for head and neck squamous cell carcinoma. • An incomplete response on PET-CT has a limited positive predictive value and optimal management is uncertain. • These data show that with a 'second-look' interval PET-CT, the majority of patients convert to a complete metabolic response. When there is doubt about clinical and radiological response, a 'second-look' PET-CT can be used to spare patients unnecessary surgical intervention

The antecedents of process integration in business process outsourcing and its effect on firm performance

As service processes become candidates for outsourcing, interest in the global business process outsourcing (BPO) industry has grown considerably. In this study, drawing on information processing theory, we examine the role of integration in BPO and its effect on BPO firm performance. BPO Integration is concerned with the overall coordination of business processes and activities across different units within the outsourced environment. It involves both internal process integration – effective integration of task execution within the BPO and external process integration – effective integration between the BPO and their clients. Using survey data gathered from 205 Indian BPO service providers, we analyze the antecedents of process integration and its impact on BPO performance. The antecedents we examine are task complexity, task security, end customer orientation of the client and IT capability of the BPO. Among other results, we find that both internal and external process integration partially mediate the impact of the antecedents on performance. We draw managerial implications of our research to practicing BPO and client managers on how BPO outsourcing can be made successful