Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Rinaldi, Gabriel; Kutschera, Ina; Bartholomae, Cynthia C.; Kalle, Christof von; Schmidt, Manfred

doi:10.3791/51543

Cited by 11 publications

(7 citation statements)

References 31 publications

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“…Next, we checked viral insertion sites using non-restrictive linear amplification-mediated PCR (nrLAM-PCR) (Figure 5B), a sensitive technique that can detect clonal insertions as discrete bands, which can then be sequenced if needed. 34 We invariably found a smear of bands indicating polyclonal hematopoiesis with very little indication of oligoclonality, except for a few minor bands from which we could not get extra specific insertion site information by sequencing. We conclude that there was no evidence of vector-induced clonal selection.…”

Section: Preclinical Release Tests Of the Vectormentioning

confidence: 78%

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Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

García-Pérez

Eggermond

Roon

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1 À/À mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34 + cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.

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Section: Preclinical Release Tests Of the Vectormentioning

confidence: 78%

“…Lentiviral insertion site was analyzed by nrLAM-PCR on murine BM DNA samples as described by Schmidt and colleagues. 34…”

Section: Nrlam-pcrmentioning

confidence: 99%

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

García-Pérez

Eggermond

Roon

et al. 2020

Molecular Therapy - Methods & Clinical Development

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“…Standard LAM-PCR assay was performed as previously described. 27,32 Briefly, LAM-PCR was initiated with two 50-cycles linear PCRs and restriction digest was performed using the MseI restriction enzyme. Two 35-cycles exponential PCRs were performed and LAM-PCR amplicons were visualized on Spreadex (Elchrom Scientific) gels to evaluate PCR efficiency.…”

Section: Methodsmentioning

confidence: 99%

Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients

et al. 2016

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Recombinant adeno-associated viral vectors (rAAV) currently constitute a real therapeutic strategy for the sustained correction of diverse genetic conditions. Though a wealth of preclinical and clinical studies have been conducted with rAAV, the oncogenic potential of these vectors is still controversial, particularly when considering liver-directed gene therapy. Few preclinical studies and the recent discovery of incomplete wild-type AAV2 genomes integrated in human hepatocellular carcinoma biopsies have raised concerns on rAAV safety. In the present study, we have characterized the integration of both complete and partial rAAV2/5 genomes in nonhuman primate tissues and clinical liver biopsies from a trial aimed to treat acute intermittent porphyria. We applied a new multiplex linear amplification-mediated polymerase chain reaction (PCR) assay capable of detecting integration events that are originated throughout the rAAV genome. The integration rate was low both in nonhuman primates and patient's samples. Importantly, no integration clusters or events were found in genes previously reported to link rAAV integration with hepatocellular carcinoma development, thus showing the absence of genotoxicity of a systemically administered rAAV2/5 in a large animal model and in the clinical context.

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“…Read-through and fusion transcripts were detected via LAM-or nrLAM-PCR. (nr)LAM-PCR, 454 sequencing, and bioinformatical data analysis were performed as described before, [30][31][32][33] with the following adjustments in the nrLAM-PCR protocol. After magnetic capture of the linear PCR product, beads were washed and resuspended in 10 lL of mastermix for linker cassette ligation using CircLigaseÔ ssDNA Ligase (Epicentre): 1.5 lL of water, 1 lL of 10 • CircLigaseÔ reaction buffer, 1 lL of ssDNA linker (10 pmol/lL), 0.5 lL of MnCl 2 (50 mM), 0.5 lL of ATP (1 mM), and 5 lL of 50% PEG 8000 (preheated for 5 min to 60°C).…”

Section: Integration Site and Chimeric Transcript Analysismentioning

confidence: 99%

Lentiviral Vector Promoter is Decisive for Aberrant Transcript Formation

et al. 2017

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Lentiviral vectors hold great promise for the genetic correction of various inherited diseases. However, lentiviral vector biology is still not completely understood and warrants the precise decoding of molecular mechanisms underlying integration and post-translational modification. This study investigated a series of self-inactivating (SIN) and full long terminal repeat (LTR) lentiviral vectors that contained different types of promoters with or without a transgene to gain deeper insights in lentiviral target site selection and potential perturbation of cellular gene expression. Using an optimized nonrestrictive linear amplification-mediated polymerase chain reaction (nrLAM-PCR) protocol, vector structure-dependent integration site profiles were observed upon transduction of mouse lin hematopoietic progenitors in vitro. Initial target site selection mainly depended on the presence of the promoter while being independent of its nature. Despite the increased propensity for read-through transcription of SIN lentiviral vectors, the incidence of viral-cellular fusion transcript formation involving the canonical viral splice donor or cryptic splice sites was reduced in both unselected primary lin cells and transformed 32D cells. Moreover, the strength of the internal promoter in vectors with SIN LTRs is decisive for in vitro selection and for the abundance of chimeric transcripts, which are decreased by moderately active promoters. These results will help to better understand vector biology and to optimize therapeutic vectors for future gene therapy applications.

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Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Cited by 11 publications

References 31 publications

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

Recombinant AAV Integration Is Not Associated With Hepatic Genotoxicity in Nonhuman Primates and Patients

Lentiviral Vector Promoter is Decisive for Aberrant Transcript Formation

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