The universal lentiviral packaging cell

Lead Research Organisation: University College London
Department Name: Immunology and Molecular Pathology

Abstract

Inherited diseases are caused by mistakes in the DNA of patients. In the case of rare inherited immunodeficiencies diseases (Baby in the Bubble syndrome), they can be cured by delivering a correct copy of the faulty DNA using an engineered virus (gene therapy). This project is to be carried out by scientists in the MRC/UCL Medical Molecular Virology Centre at UCL. They will develop a safer and more efficient method to produce engineered viruses for clinical gene therapy trials.

Technical Summary

Lentiviral vectors integrate and express transgenes in most cells. Due to cytotoxicity of viral proteins vectors are currently produced by transient transfection. This is costly, laborious, inherently unsafe and hard to scale-up. We recently reported a prototype packaging cell that continuously produced high-titre lentiviral vectors (see key reference). We now request funding to produce a safe, easily manipulated packaging system suitable for clinical lentiviral vector production by the following strategy, using recombination mediated cassette exchange:
1. Infect 293T cells with an MLV vector containing a loxP site in LTR, then transient transfection of Cre recombinase and selectable lentiviral Gag-Pol cassettes.
2. Infect these cells with a second MLV vector containing an FRT site, then transfected with Flp recombinase and selectable lentiviral vector cassettes.
3. Gammaretroviral envelopes will be expressed, viral safety and immunogenicity will be tested.

Publications


10 25 50
Anderson I (2014) Heat shock protein 90 controls HIV-1 reactivation from latency. in Proceedings of the National Academy of Sciences of the United States of America
Berkhout B (2013) Obituary: Kuan-Teh Jeang. in Retrovirology
 
Description Biomedical Resource
Amount £2,000,000 (GBP)
Organisation German Federal Ministry of Education and Research 
Sector Public
Country Germany, Federal Republic of
Start 01/2008 
End 12/2012
 
Description EU FP7 HEALTH 2012 INNOVATION-1 (STAUSS)
Amount € 6,000,000 (EUR)
Organisation Community Research and Development Information Service (CORDIS) 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start  
End 10/2015
 
Description UCLH BRC Funding for Wolfson Gene Therapy Unit, 2013-2016 (M. Collins)
Amount £520,000 (GBP)
Organisation Hungarian Academy of Sciences (MTA) 
Department Biological Research Centre (BRC)
Sector Academic/University
Country Hungary, Republic of
Start 01/2013 
End 01/2016
 
Title Lentiviral packaging cell 
Description In order to make large ammounts of lentiviral vectors for clinical use, a stable cell line production system is necessary. In this project we have made a GMP compatible cell line expressing higher levels of HIV gag-pol than previously reported by our group and others. We are now completing the work. 
Type Of Material Cell line 
Provided To Others? No  
Impact Too soon. These cells will be useful when we finish them 
 
Description Collaboration with Adrian Hill and Helen McShane Oxford supported by UCL CBRC grant (COLLINS) 
Organisation University of Oxford
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Lentiviral vectors
Collaborator Contribution Novel adjuvant molecules, TB antigens and models
Impact in progress
Start Year 2011
 
Description Dennis Burton Lab Visit (MCCOY) 
Organisation Scripps Research Institute
Country United States of America 
Sector Charity/Non Profit 
PI Contribution This travel grant was used to accelerate a HIV antigen development project
Collaborator Contribution This travel grant was used to accelerate a HIV antigen development project
Impact not known yet
Start Year 2013
 
Description Discovery of new cellular targets for antiretroviral drug development (FASSATI) 
Organisation Biodim Laboratory
Country France, French Republic 
Sector Private 
PI Contribution Developed a novel cell-based, high through put screening assay. Discovered a new cell target important for HIV-1 infection.
Collaborator Contribution Provided chemical library of 10,000 compounds to perform screening and cherry picked compounds.
Impact Eleven new hits identified to be followed up
Start Year 2012
 
Description Discovery of new cellular targets for antiretroviral drug development (FASSATI) 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Developed a novel high through put screening, cell-based assay. Discovered a new cell target important for HIV
Collaborator Contribution Provided novel lead compounds that inhibit the target identified in our high through put screening
Impact in the process of testing the compounds
Start Year 2013
 
Description Dr Marcus Dorner, Imperial College 
Organisation Imperial College London (ICL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided data and expertise on the effect of Hsp90 on regulation of HIV-1 latency, provided specific Hsp90 inhibitor.
Collaborator Contribution Humanized mouse models for HIV-1 infection to test the effect of selective Hsp90 inhbitors on HIV-1 latency.
Impact experiments ongoing
Start Year 2014
 
Description Dr Peter Cherepanov, CRUK 
Organisation Cancer Research UK (CRUK)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution provision of expression constructs recomninat protein expression and methods for immunoprecipitation. Expertise on nuclear transport receptors.
Collaborator Contribution Structural biology of transportin 3.
Impact One paper in PNAS
Start Year 2010
 
Description Dr Richard Benarous, Mutabilis, France 
Organisation University College London (UCL)
Department Metbolism and Endocrinology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Performed novel high through put screening to identify host factos necessary for HIV-1 replication.
Collaborator Contribution Provided expertise and library of 10,000 small chemical compounds selected from a 150,000 compounds library.
Impact Identified four new compounds with antiretroviral activity: they belong to a new class of antiretrovirals .
Start Year 2012
 
Description Dr. Ian Taylor, MRC NIMR, Mill Hill (FASSATI) 
Organisation Medical Research Council (MRC)
Department Biomedical NMR Centre at NIMR
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided data and expertise on the role of the antibiotic Coumermycin-A1 in blocking HIV-1 replication
Collaborator Contribution co-crystal structure of Coumermycin-A1 and HIV-1 capsid protein
Impact one paper in preparation
Start Year 2012
 
Description Hsp90 controls HIV-1 reactivation from latency (FASSATI) 
Organisation International Centre for Genetic Engineering and Biotechnology (ICGEB)
Country Italy, Italian Republic 
Sector Charity/Non Profit 
PI Contribution Discovered that Hsp90 is required for HIV-1 gene expression and reactivation from latency, described the pathway involved.
Collaborator Contribution Discovered that hyperthermia stimulates HIV-1 replication and reactivation. Contributed imaging methods to visualise nuclear migration of Hsp90 and other components.
Impact One paper in PLoS Path (2012), one invited review in Parasitology (to be submitted shortly), one manuscript submitted and one MRC grant application submitted in September 2013.
Start Year 2011
 
Description Hsp90 controls HIV-1 reactivation from latency (FASSATI) 
Organisation Pasteur Institute, Paris
Country France, French Republic 
Sector Academic/University 
PI Contribution Discovered that Hsp90 is required for HIV-1 gene expression and reactivation from latency, described the pathway involved.
Collaborator Contribution Discovered that hyperthermia stimulates HIV-1 replication and reactivation. Contributed imaging methods to visualise nuclear migration of Hsp90 and other components.
Impact One paper in PLoS Path (2012), one invited review in Parasitology (to be submitted shortly), one manuscript submitted and one MRC grant application submitted in September 2013.
Start Year 2011
 
Description Hsp90 controls HIV-1 reactivation from latency (FASSATI) 
Organisation University College London (UCL)
Department UCL Division of Biosciences
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Discovered that Hsp90 is required for HIV-1 gene expression and reactivation from latency, described the pathway involved.
Collaborator Contribution Discovered that hyperthermia stimulates HIV-1 replication and reactivation. Contributed imaging methods to visualise nuclear migration of Hsp90 and other components.
Impact One paper in PLoS Path (2012), one invited review in Parasitology (to be submitted shortly), one manuscript submitted and one MRC grant application submitted in September 2013.
Start Year 2011
 
Description Llama VHHantobody fragments to neutralise viruses (WEISS) 
Organisation University College London (UCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution virology
Collaborator Contribution VHH technology
Impact Research papers and 1 patent filed
Start Year 2006
 
Description Mechanism of viral transport across nuclear pores (FASSATI) 
Organisation University College London (UCL)
Department London Centre for Nanotechnology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Expertise on nucleo-cytoplasmic trafficking of viruses and large molecules.
Collaborator Contribution Atomic force microscopy and physical modelling of nuclear pores.
Impact One modelling paper (Phys. Rev. E. 2012), one review (Soft Matter 2013), one paper to be submitted shortly.
Start Year 2011
 
Description Novel broadly neutralizing Llama VHH (FASSATI) 
Organisation University College London (UCL)
Department UCL Division of Infection & Immunity
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Developed and performed high through put screening to identify novel broadly neutralizing LLama VHH, identified three new VHH.
Collaborator Contribution Characterised VHH, area of binding, performed deep sequencing to demonstrate that novel VHH arised after immunization.
Impact One paper to be submitted shortly.
Start Year 2012
 
Description Population dynamics of T cell responses analysed using high-throughput sequencing of TCR repertoire (CHAIN) 
Organisation The Hebrew University of Jerusalem
Department Weizmann Institute of Science
Country Israel, State of 
Sector Academic/University 
PI Contribution Developemnt of software for seqeunce analysis.
Collaborator Contribution Immunisations and seqeuncing
Impact Still in progress
Start Year 2011
 
Description Prof. Olivier Schwartz, Pasteur Institute, Paris (FASSATI) 
Organisation Pasteur Institute, Paris
Country France, French Republic 
Sector Academic/University 
PI Contribution Provided data and expertise on the role of Hsp90 in HIV-1 infection
Collaborator Contribution Provided data and expertise on mechanisms contributing to enhanced HIV-1 replication during hyperthermia (fever)
Impact One paper in PLoS Pathogens describing the effect of fever on HIV-1 replication and the role of Hsp90
Start Year 2011
 
Description Professor Benny Chain, UCL and MRC Centre 
Organisation University College London (UCL)
Department MRC UCL Centre for Medical Molecular Virology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided data and expertise on nuclear import of DNA.
Collaborator Contribution Provided data and expertise on biophysical properties of transfected DNA and on innate Immunity.
Impact One MSc Thesis (Tse Wang) and one paper published in the journal Traffic
Start Year 2011
 
Description Professor Charles Bangham, Imperial College 
Organisation Imperial College London (ICL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Discovered new host factor involved in HIV-1 replication that affects integration site selection.
Collaborator Contribution High through put sequencing of HIV-1 integration sites.
Impact One paper in preparation, one grant application submitted to MRC.
Start Year 2012
 
Description Professor Christopher Moody and Dr Russell Kitson, University of Nottingham 
Organisation University of Nottingham
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided data and expertise on the role of Hsp90 in HIV-1 latency.
Collaborator Contribution Provided new chemical compounds inhibitors of Hsp90
Impact One paper in PNAS
Start Year 2012
 
Description Professor Mike Stratton and Dr Elisabeth Murchison, Wellcome Trust Sanger Institute 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Charity/Non Profit 
PI Contribution Provided data and expertise on the canine Venereal Transmissible Tumour (CTVT).
Collaborator Contribution Provided data and expertise on the genome of CTVT
Impact A paper describing the CTVT genome published in Science, another publication on the tumour transcriptional changes is in preparation.
Start Year 2010
 
Description Professor Olivier Schwartz, Pasteur Institute, Paris 
Organisation Pasteur Institute, Paris
Country France, French Republic 
Sector Academic/University 
PI Contribution Provided data and expertise on the role of Hsp90 in HIV-1 infection.
Collaborator Contribution Provided data and expertise on mechanisms contributing to enhanced HIV-1 replication during hyperthermia (fever).
Impact One paper in PLoS Pathogens describing the effect of fever on HIV-1 replication and the role of Hsp90
Start Year 2011
 
Description Raymond Welsh Lab Visit (PEPPA) 
Organisation University of Massachusetts (UMass)
Country United States of America 
Sector Academic/University 
PI Contribution address how T cell regulation by NK cells contribute to overall disease pathogenesis
Collaborator Contribution development of valuable collaboration, also link with a planned project on regulatory potential of NK cells in HBV with the MRC unit in the Gambia
Impact not yet known
Start Year 2013
 
Description Super-resolution imaging: probe development (GROVE) 
Organisation University College London (UCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Project conception, development and application
Collaborator Contribution Project conception, development and application
Impact not yet known
Start Year 2013
 
Description UCL Gene Therapy Consortium 
Organisation University College London (UCL)
Department UCL Biosciences
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We are developing reagents and processes for clinical lentiviral vector production.
Collaborator Contribution Provision of GMP viral production facilities
Impact We have set up an MHRA accredited GMP production facility
Start Year 2008
 
Description Understanding the coordination of hepatitis C virus entry and humoral immune evasion (GROVE) 
Organisation University College London (UCL)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Project conception, development and application
Collaborator Contribution Access to clinical samples
Impact not yet known
Start Year 2013
 
Description Veronique Birault, GSK, Stevenage. 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Provided data on a new host factor to be used as an antiretroviral target.
Collaborator Contribution Provided lead compounds that target the new host factor discovered by us.
Impact In the process of performing SAR studies with GSK compounds
Start Year 2013
 
Description Wuerzburg Collaboration (STAUSS) 
Organisation University of Würzburg
Country Germany, Federal Republic of 
Sector Academic/University 
PI Contribution TBA
Collaborator Contribution TBA
Impact TBA
Start Year 2009
 
Description Carolton Primary School Visit 2012 (TOWERS/MILNE) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact 40 local school children attended open day in the Centre labs; consisted of interactive introductory talk, followed by lab rotations where pupils preformed DNA extractions, microscope work, pipetting skills, artwork. Pupils asked many questions and the Centre received thank you letters expressing their increases interest in science and had a unique opportunity to do real lab-based tasks.
Year(s) Of Engagement Activity 2012
 
Description Contribution to the Financial Times "New synthetic life needs context and regulation"(A. Fassati) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact to be added
Year(s) Of Engagement Activity 2010
URL http://www.ft.com/cms/s/0/3da6db20-6796-11df-a932-00144feab49a.html#axzz2hsOEkNfY
 
Description HIV & AIDS (WEISS) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact too soon to say
Year(s) Of Engagement Activity 2013
 
Description Institute of Immunity and Transplantation Opening Symposium (GROVE) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The symposium was filmed and I worked with a video editor to create a digital version of the presentation for You Tube
Year(s) Of Engagement Activity 2013
 
Description Lecture "Conscience and values in the response to infectious diseases", Thomas More Institute, London. (A. Fassati) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact to be added
Year(s) Of Engagement Activity 2011
URL http://thomasmoreinstitute.org.uk/papers/conscience-and-values-in-the-response-toinfectious-diseases...
 
Description Lecture on the AIDS epidemic for the charity "Mothers to Mothers" London. (A. Fassati) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact to be added
Year(s) Of Engagement Activity 2011
URL http://www.m2m.org
 
Description Lecture to Y6 children "Investigating microbes" at the Oratory RC Primary School, London. (A. Fassati) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact to be added
Year(s) Of Engagement Activity 2014
 
Description Live comment on the origin of the AIDS epidemic on Arise News (A. Fassati) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact to be added
Year(s) Of Engagement Activity 2014
URL http://www.arise.tv/arise-news-show/africa-wrap-03-10-8501
 
Description Lunchtime Lecture UCL (COLLINS) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 800 You tube hits
Year(s) Of Engagement Activity 2012
 
Description MRC@UCL Centenary Science Fair (ALL MEMBERS) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 500+ members of the public (families, adults, school children) attended a science fair held @ UCL, Centre members contributed 3 hands-on workshop stalls, and presented separate talks. The public were given opportunities to perform tasks associated with the work of the Centre, and asked questions/contributed to discussions about what the Centre does and general science queries. Feedback included saying children had increased interest in developing their science education and awareness of virology principles.
Year(s) Of Engagement Activity 2013
 
Description Mayflower & Gayhurst Primary School visits 2013 (TOWERS/MILNE) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Centre members visited local schools and presented an interactive talk about virology. Pupils asked many questions and the Centre received thank you letters expressing their increases interest in science and had a unique opportunity to do real lab-based tasks.
Year(s) Of Engagement Activity 2013
 
Description Mayflower & Gayhurst Primary schools visits 2012 (TOWERS/MILNE) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Centre members visited local schools and presented an interactive talk about virology. Pupils asked many questions and the Centre received thank you letters expressing their increases interest in science and had a unique opportunity to do real lab-based tasks.
Year(s) Of Engagement Activity 2012
 
Description Public lecture (STAUSS) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Approximately 200 lay people attended lecture on T Cell therapy

Lecture was recorded and is available on You-tube
Year(s) Of Engagement Activity 2012
 
Description School Visit Primrose Hill (STAUSS) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact Parents attended a presentation of our research and all children attended an assembly

money raised for cancer research
Year(s) Of Engagement Activity 2012
 
Description Talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Participants in your research and patient groups
Results and Impact I have talked about this work at several national and international gene therapy meetings and posters have been presented

applications to work in the lab, PhD student recruitment
Year(s) Of Engagement Activity 2007,2008,2009
 
Description Talk in Bloomsbury Festival (M COLLINS) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Cosy Science, "viruses are our friends", The Marquis of Cornwallis pub, 2013 for 50+ local residents

raised public awareness
Year(s) Of Engagement Activity 2013