Stable and Predictable Lentiviral Vector Production at Clinical Scale.

SIN Retroviral Vectors Expressing COL7A1 Under Human Promoters for Ex Vivo Gene Therapy of Recessive Dystrophic Epidermolysis Bullosa

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro and in animals. Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression. The packaging signal (Ψ) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (http://www.addgene.org/). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time. Commercially available transfection kits may support high efficiency of transfection, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production. In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10. Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (Ψ) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (http://www.addgene.org/). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production. In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

Mechanism of Reduction in Titers From Lentivirus Vectors Carrying Large Inserts in the 3′LTR

Genotoxic Potential of Lineage-specific Lentivirus Vectors Carrying the β-Globin Locus Control Region

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

68. Improved lentiviral vector system with enhanced safety features

Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element

On Target: New Envelopes for Lentiviral Vectors

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN gamma-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN gamma-retroviral vectors for clinical trials.

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.

<b>Elisa Manzotti</b>, Founder, BioInsights, speaks to <b>Hanna Leinonen, John Moscariello, Lee DaviesScott Cross </b>and <b>Will Junker</b> <div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/H Leinonen copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Hanna Leinonen</h3><h5 class="author-title">Senior Scientist, Kuopio Center for Gene and Cell Therapy</h5> Hanna M. Leinonen holds a PhD in Molecular Medicine from the University of Eastern Finland (2014). She has 15 years’ experience on lentiviral vectors and 6 years’ experience on bioprocessing. She is currently working as a Senior Scientist in Kuopio Center for Gene and Cell Therapy (Finland) focusing on development of upstream processing for viral vectors (lentivirus, adenovirus, AAV). </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/26118d9d52f34a12859025a3d5f4aabc.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">John Moscariello</h3><h5 class="author-title">Executive Director, Viral Vector and Gene Editing Process Development, Bristol Myers Squibb</h5> John Moscariello currently serves as Executive Director of Viral Vector and Gene Editing Process Development at Bristol-Myers Squib (BMS). At BMS, John’s group develops viral vector processes that enable rapid timelines to generate clinical viral vectors as well as the development and characterization of commercial viral vector processes. Prior to his work at BMS, John was the Vice President of Process Development at AGC Biologics where his team was responsible cell line development, upstream and downstream process development, analytical and formulation development, and technical support for AGC Biologic’s commercial manufacturing facility and supported development activities from generating processes for Toxicology/Phase 1 supply up to and including commercialization and post-approval process support. John is very active in the biotechnology community. He is on the Scientific Advisory Board for various conferences, including the BioProcess International conference series, and the CBI conference series on achieving efficient facilities and the next frontier of single-use technologies. John obtained his Ph.D. in Chemical and Biological Engineering from the University of Wisconsin-Madison, and his B.Eng. in Chemical Engineering from the University of Delaware. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Lee Davies 1.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Lee Davies</h3><h5 class="author-title">Director, Process Research &amp; Development, Oxford Biomedica</h5><p class="oneComWebmail-MsoNormal"><i>As Director of Process Research and Development, Lee helps drive development, innovation and improvements in&nbsp;</i><i>world-class lentiviral vector manufacturing processes,&nbsp;</i><i>applying scientific and engineering principles to all aspects of upstream and downstream process development, including process optimisation and scale-up, technology transfer to GMP manufacturing and process characterisation to support chemistry, manufacturing and controls (CMC) regulatory submissions.&nbsp;</i><i>As part of&nbsp;the</i><i>&nbsp;Process Research and Development team, Lee works</i><i>&nbsp;at the&nbsp;cutting edge of gene therapy, ensuring its applicability to the needs of the wider gene and cell therapy community. The team’s mission covers the&nbsp;</i><i>life cycle of gene therapies, taking</i><i>&nbsp;processes from the early phase development work through to&nbsp;fully optimised large scale, GMP-ready processes, manufacturing and commercialisation.&nbsp;</i><i>Prior to joining Oxford Biomedica 6 years ago, Lee worked at the University of Oxford as a postdoctoral researcher and obtained a D.Phil in Gene Therapy for Cystic Fibrosis.</i> </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Scott_122321_WebReady copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Scott Cross</h3><h5 class="author-title">Senior Principal, Dark Horse Consulting</h5> Scott has over 20 years experience working with viral vectors, vaccines, and biologics in GMP environments. He has been responsible for cleanroom design, build out and commissioning of vector facilities, as well as the oversight of viral vector, cell therapy, fill finish and manufacturing support departments. Scott started his career at Merck and Co. where he worked on the development of an Adenovirus-based HIV vaccine and later, the development, optimization, and validation of release assays for live virus vaccines. He then moved to Indiana University (IU) where he managed the IU Vector Production Facility (IUVPF), overseeing GMP production and testing of Lentiviral and Retroviral vectors for the IUVPF and the National Gene Vector Laboratories (NGVL). While at the IUVPF he also managed the design, build and commissioning of a new viral vector GMP production and testing facility. After leaving the IUVPF, Scott joined Cincinnati Children’s Hospital Medical Center (CCHMC) managing the GMP Vector Production Facility, Viral Vector Core, and the Aseptic Processing Labs. Scott then moved to the University of Florida where he was the Director of Cell Therapy, Fill Finish and Manufacturing Support Operations for Florida Biologix and Brammer Bio. He was also responsible for the design of two new fill finish suites. Most recently, Scott served as Vice President of Vector Operations at Orchard Therapeutics where he was one of the original ten members and responsible for viral vector development, GMP vector production, plasmid production, oversight of 10 CDMOs, due diligence, and facility design. </div></div></div><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://cdn.insights.bio/uploads/Will Junker headshot copy.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">Will Junker</h3><h5 class="author-title">Head of Vector Manufacturing Quality, Kite Pharma</h5> Will Junker is Head of Vector Quality and oversees the operation of QC, QA and QE to bring Kite’s vector manufacturing facility on line through licensure. Before this role, Will built Kite’s Quality Engineering function globally. His 35 year career in BioPharma includes 13 years at AstraZeneca and 15 years at Aventis/Sanofi as well as a few years at Baxter BioScience in technical/quality roles of increasing responsibility. </div></div></div>