VISMapper: ultra-fast exhaustive cartography of viral insertion sites for gene therapy

AbstractThe integration of a viral genome into the host genome has a major impact on the trajectory of the infected cell. Integration location and variation within the associated viral genome can influence both clonal expansion and persistence of infected cells. Methods based on short-read sequencing can identify viral insertion sites, but the sequence of the viral genomes within remains unobserved. We develop PCIP-seq, a method that leverages long reads to identify insertion sites and sequence their associated viral genome. We apply the technique to exogenous retroviruses HTLV-1, BLV, and HIV-1, endogenous retroviruses, and human papillomavirus.

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Abstract 257: Bone Marrow Cells Form New Cardiomyocytes in the Infarcted Myocardium

Circulation Research ◽

10.1161/res.113.suppl_1.a257 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Fumihiro Sanada ◽

Junghyung Kim ◽

Anna Czarna ◽

Sergio Signore ◽

Andrea Sorrentino ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Fluorescent Proteins ◽

Infarcted Myocardium ◽

Multiple Cell ◽

Insertion Sites ◽

Color Combinations ◽

Viral Insertion ◽

Marrow Cells

The ability of c-kit-positive bone marrow cells (c-kit-BMCs) to form cardiomyocytes following delivery to the acutely infarcted myocardium is a matter of debate. In an attempt to resolve the controversy, we raised the possibility that c-kit-BMCs represent a functionally heterogeneous pool, containing cells with distinct transdifferentiation potential. To test this hypothesis, c-kit-BMCs were infected with EGFP-lentiviruses and injected in infarcted hearts. At 1 and 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells (ECs), fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for unique sites of viral insertion in the infected c-kit-BMCs and their progeny. Common insertion sites were found in the DNA of all cell populations, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, the 31 detected clones shared 9 distinct sites of integration, suggesting that myocytes in different animals were generated by specific subsets of c-kit-BMCs, which retained the ability to transdifferentiate. To strengthen the observation that myocardial regeneration is oligoclonal in nature, freshly isolated FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors resulted in the formation of numerous c-kit-BMC clones of mixed colors. RGB-infected HSCs were injected in infarcted rats; at 4 days, engrafted c-kit-BMCs showed a polyclonal pattern characterized by the presence of cells labeled by most of the color combinations seen in vitro. However, at 2 weeks, only a few of the homogeneously colored colonies seen at 4 days persisted and formed large patches of cells composed of myocytes, ECs, and smooth muscle cells. Our findings document that c-kit-BMCs are functionally heterogeneous and have different ability to survive, engraft, and transdifferentiate in the ischemic heart. At present, clonal c-kit-BMCs are being analyzed by RNA-sequencing for the identification of the unique phenotypical properties of c-kit-BMCs with high transdifferentiation potential.

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Viral Insertion Site Detection and Analysis in Cancer Gene Therapy

Gene Therapy of Cancer ◽

10.1016/b978-0-12-394295-1.00003-2 ◽

2014 ◽

pp. 35-46 ◽

Cited By ~ 1

Author(s):

Hua Fung ◽

Stanton L. Gerson

Keyword(s):

Gene Therapy ◽

Insertion Site ◽

Cancer Gene Therapy ◽

Cancer Gene ◽

Viral Insertion

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Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy

Genome Biology ◽

10.1186/gb-2007-8-s1-s12 ◽

2007 ◽

Vol 8 (Suppl 1) ◽

pp. S12 ◽

Cited By ~ 37

Author(s):

Christopher S Hackett ◽

Aron M Geurts ◽

Perry B Hackett

Keyword(s):

Gene Therapy ◽

Functional Genomics ◽

Insertion Sites ◽

Dna Vector

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Insertion Sites in Engrafted Cells Cluster Within a Limited Repertoire of Genomic Areas After Gammaretroviral Vector Gene Therapy

Molecular Therapy ◽

10.1038/mt.2011.178 ◽

2011 ◽

Vol 19 (11) ◽

pp. 2031-2039 ◽

Cited By ~ 30

Author(s):

Annette Deichmann ◽

Martijn H Brugman ◽

Cynthia C Bartholomae ◽

Kerstin Schwarzwaelder ◽

Monique MA Verstegen ◽

...

Keyword(s):

Gene Therapy ◽

Insertion Sites

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HGT-ID: an efficient and sensitive workflow to detect human-viral insertion sites using next-generation sequencing data

BMC Bioinformatics ◽

10.1186/s12859-018-2260-9 ◽

2018 ◽

Vol 19 (1) ◽

Author(s):

Saurabh Baheti ◽

Xiaojia Tang ◽

Daniel R. O’Brien ◽

Nicholas Chia ◽

Lewis R. Roberts ◽

...

Keyword(s):

Next Generation Sequencing ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Insertion Sites ◽

Viral Insertion ◽

Generation Sequencing

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Expression of Two Foreign Genes from the Optimal Insertion Sites of Newcastle Disease Virus Vector for Use as a Multivalent Vaccine and Gene Therapy Vector

Proceedings ◽

10.3390/proceedings2020050009 ◽

2020 ◽

Vol 50 (1) ◽

pp. 9

Author(s):

Lei He ◽

Zhenyu Zhang ◽

Qingzhong Yu

Keyword(s):

Gene Therapy ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Fluorescent Protein ◽

Transcription Unit ◽

Lower Amount ◽

Foreign Gene ◽

Entry Site ◽

Insertion Sites

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and gene therapy purposes. A majority of these NDV vectors express only a single FG or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses to express two FGs, green fluorescent protein (GFP) and red fluorescent protein (RFP) genes, from the identified optimal insertion sites, through a combination of the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches. Biological assessments showed that these recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers than those containing a single FG. The FG expression efficiencies from two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expression of FGs through the ITU approach was approximately 4-fold more efficient than that through the IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for the expression of a higher amount of FG products, whereas the IRES tactic might be useful when a lower amount of FG products are needed.

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210. Modelling Viral Insertion Sites Via Network Based Frameworks

Molecular Therapy ◽

10.1016/s1525-0016(16)34545-2 ◽

2013 ◽

Vol 21 ◽

pp. S81

Keyword(s):

Insertion Sites ◽

Viral Insertion

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Viral Insertion Safety in Patients with Glioblastoma Who Received a Novel Lentiviral MGMT-P140K Gene Therapy to Protect Bone Marrow from Chemotherapy: No Dominant Clonal Evolution Observed with Chemo-Selection

Blood ◽

10.1182/blood.v124.21.4801.4801 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4801-4801

Author(s):

Hua Fung ◽

Andrew E Sloan ◽

Jane Reese ◽

Basem M. William ◽

Karen Lingas ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Lentiviral Vector ◽

Lymphoblastic Leukemia ◽

Insertion Site ◽

Retroviral Vectors ◽

Insertion Mutagenesis ◽

Drug Selection ◽

Insertion Sites ◽

Viral Insertion

Abstract INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny. METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy. HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection. RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished. CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response. Disclosures Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.

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Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Blood ◽

10.1182/blood.v110.11.3744.3744 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3744-3744

Author(s):

Thomas R. Bauer ◽

Mehreen Hai ◽

Rima L. Adler ◽

James M. Allen ◽

Laura M. Tuschong ◽

...

Keyword(s):

Gene Therapy ◽

Viral Vectors ◽

Insertional Mutagenesis ◽

Leukocyte Adhesion ◽

Severe Combined Immunodeficiency ◽

Insertion Site ◽

Leukocyte Adhesion Deficiency ◽

Hematopoietic Stem ◽

Insertion Sites ◽

Immunodeficiency Disease

Abstract Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

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