Lentiviruses - Not just Retro Chique

    18/03/2018

    Photo by Andrew Renneisen, Philly.com: Carl June in 2015, reflecting on several years of T-cell therapy trials.

    When I first heard about the use of modern lentiviruses in clinical trials I was excited. It was early 2015 and I was searching for Philadelphia area professors to come to talk at a little science outreach festival, “Pint of Science” (now Taste of Science). One of my co-organizers of the festival announced that Carl June had agreed to come and speak at our bar venue. I quickly looked up his research and found out he was a pioneer in the use of lentiviruses to transduce T-cells for the treatment of a rare form of leukemia. He was using a relatively novel innovation called CAR-T cell therapy and had been featured in a documentary on PBS. The fact that I was also using lentiviruses in my research albeit at a much more basic level to do more basic research added to my fascination. Unfortunately that year I got sick on the day of the talk so I missed out on hearing June’s story and meeting the man. Last year, as I flew into Iowa to start at my current gene therapy job, I heard about FDA’s approval of the first CAR-T cell therapy. In just a few years gene therapy has gone from a little-known delivery technique (with a questionable safety history) to a verifiable cure for serious genetic diseases. This week I will introduce some background about lentiviruses, the vector that has enabled treatments like CAR-T cell therapy.

    What are lentiviruses?

    Lentiviruses are part of a larger genus of retroviruses that include the famous HIV (human immunodeficiency virus), which causes AIDS. Lentiviruses have been designed with safety features after years of trial and error. They are able to infect, replicate and integrate in non dividing cells, lending to their usefulness in fields of study like neuroscience and cancer immunology, where they can be used for non dividing cells such as neurons and certain macrophages.

    Cartoon depiction of a typical retrovirus / lentivirus (image courtesy of Labome):

    Lentiviral particles are spherical or pleomorphic and are 80-100 nm diameter. They contain (+) sense RNA single strand genetic material within nucleocapsids, surrounded by envelopes that wrap around the core and glycoprotein spikes that allow particles to bind to specific host cell receptors. Like all retroviruses, lentivirus capsids contain reverse transcriptase, integrase and proteases required for viral infection. Once the virus enters the host cell the RNA (+) strand is copied into double stranded DNA by reverse transcriptase and sent into the nucleus where it integrates with the host DNA (using integrase). This becomes the provirus. The viral genes are transcribed by RNA Polymerase 2, translated into viral capsid proteins and assembled into a virion. It uses the host cell membrane to package the viral RNA genome before lysing the host cell and budding off.

    Retrovirus replication cycle (image courtesy of Labome):

    viruscycle

    Up to a third of all viral vectors currently used in gene therapy are retroviruses, with lentiviruses occupying a big proportion. Four famous genes are encoded by lentiviruses: gag (group specific antigen), pro (protease), pol (polymerase) and env (envelope). These genes give rise to the viral structure and function. There are also two LTRs (long terminal repeats) which help drive gene expression, reverse transcription and integration into the host cell.

    Lentiviral sequences have undergone several design iterations since their original discovery, leading to three generations with increasing safety profiles. First generation lentiviruses were produced in large quantities by packaging cells and could be collected to be used to infect non dividing cells such as rat brain neurons. Second generation lentiviruses had parts of the HIV virulence sequences removed from the packaging construct. This system used a single packaging plasmid encoding gag, pol, rev and tat genes. Third generation lentiviruses are used in most places today. They have the tat promoter removed and have a self-inactivating design to further get rid of pathogenic features (or to reduce replication-competent recombinant viruses). The packaging system is split into a transfer plasmid, packaging plasmids and envelope plasmid. Despite these safety enhancements lentiviruses still retain many identical features to HIV in order to maintain transduction efficiency. Many of these are based on other animals, such as SIV (simian immunodeficiency virus), FIV (feline immunodeficiency virus), BIV (bovine immunodeficiency virus) and EAIV (equine immunodeficiency virus).

    Second Generation Lentiviral gene plasmid vs Third generation lentiviral gene plasmid (image courtesy of Addgene):

    Addgene

    Manufacturing
    Lentiviruses are primarily made by transient transfection of a packaging cell line. That means transfecting cells with the three plasmids encoding transfer genes, packaging genes and envelope genes together and waiting 24-72 hours for cells to release the virus.

    Viral production scheme (image courtesy of Addgene):

    Addgene

    Available packaging cell lines (Image courtesy of Inotech.com):

    Packaging cell lines are based on human 293 cells containing oncogenes, such as SV40 large T antigen (293T cells). These cells divide rapidly on fixed surfaces or in bioreactors. However, for clinical development, human cell lines have safety risks since they can generate replicative-competent particles through homologous recombination. There can also be human pathogens that cause contamination. Thus, a variety of cell lines from other species can be obtained. There are also disadvantages to using non-human cells, such as inadequate glycosylation of proteins, leading to a low quality virus.

    Advantages and Disadvantages of using different cell lines to grow lentiviruses (Image courtesy of Temple University, Bioprocess Basics class):

    A variety of different cell culture systems exist and they can be chosen according to the stage of clinical development. For general viral vector gene therapy development, adherent cultures are usually used for early development, small scale production while suspension cultures are necessary for larger scale production. However for lentiviral vector manufacture, even clinical batches often rely on adherent, disposable systems, such as T-flasks, multi-tray factories and roller bottles. These culture systems can produce between 10-40 Liters of vector under GMP conditions, required for clinical trials. Any cell line that can produce 10e7 infectious units per ml are considered adequate for scaling up. If cell culture systems need to be scaled up in future bioreactors will need to come into play. These can hold hundreds or thousands of liters depending on requirements.

    Different cell culture systems:

    When it comes to defining the quality of a viral vector product there are a number of factors to consider. FDA guidelines for Good Manufacturing Practices (GMPs) were designed along with the International Conference on Harmonization (ICH) guidelines to ensure quality and safety are built into the product and the process of manufacturing. Everything done in a manufacturing space by modern day drug and biological companies must follow Quality Guidelines. The most important set of these are:

    ICH Q8 - Pharmaceutical Development
    ICH Q9 - Quality Risk Management
    ICH Q10 - Pharmaceutical Quality Systems
    ICH Q11 - Development and Manufacture of Drug Substances

    In lentiviral manufacture, one of the keys to complying with these guidelines is to define the Critical Quality Attribute parameters that are appropriate for the cell culture system. Such parameters include pH, temperature, osmolarity, sugar carbon source, oxygen, carbon dioxide concentrations and what type of substrate the cells attach to. Cholesterol content and protein glycosylation levels are often most closely monitored to test viral vector stability since these are the first attributes to change during degradation. The greatest disadvantage of lentiviral vectors are their low titers (only 10e6-10e7 compared to AAV (>10e12)), short half-life (8-12 hours) and low ratios of infectious particles compared to total particles. Lentiviruses also rely on culture media that contains up to 10% animal sera, which adds risk to contamination of the final product and requires an increased level of monitoring.

    Clinical Applications

    The first clinical trials using an unsafe retrovirus, Murine Leukemia Virus (MLV), occurred back in 2000 when doctors in Paris tried to treat children with Severe Combined Immunedeficiency (SCID)-XI disease. After initial signs of success it was discovered that these children developed leukemia-like disorder. The virus had transduce T-cells at the wrong site close to an oncogene and after several rounds of cell division this resulted in cancer.

    Recent clinical trials use third generation lentiviruses and they have now been proven to be safe and effective in immunotherapy. Most notably, they have been used to transduce lymphocytes with T cell receptors or chimeric antigen receptors (CAR). In 2011 the first published success of CAR-T cell therapy came out from the Carl June lab, showing significant improvements for three patients with chronic lymphocytic leukemia. Other clinical trials have been using lentiviruses to transduce hematopoietic stem cells to correct rare genetic diseases such as X-linked adrenoleukodystrophy, Wiskott-Aldrich syndrome and haemoglobinopathies. These trials have not gone on without failure and in each case a number of patients did not see improvement of their symptoms or have even had relapses in disease. However, with the advent of genome editing technology such as TALENs, Cas9 and CRISPR guide RNAs, lentiviruses will play a potentially powerful role in providing effective delivery to precisely target faulty genes.

    CAR-T cell therapy schematic:

    CARTcell

    Last year, with the FDA approval of Yescarta, the first CAR-T cell therapy for non-Hodgkin lymphoma and Kymriah, the first gene therapy for acute lymphoblastic leukemia, it seems gene therapy has truly gone full circle. Retroviruses have now made a come-back as the trendy therapeutic innovation of our time, shedding their old image of the ethically and technically dangerous rogue technology. Scientific, medical and patient communities around the world are now waiting with baited breathe to see if follow-up studies for early trials for these newly approved treatments yield equally desirable results years down the line. In the mean time companies the world over will be scrambling to get in on the new Gold Rush of gene therapy.

     

    References:

    http://www.philly.com/philly/blogs/healthcare/Penn-CHOP--Promising-new-results-from-immunotherapy.html

    Naldini et al. Lentiviral vectors, two decades later. Science. 353(6304): 1101-1102 (2016).

    Hacein-Bey-Abina, S. et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N. Engl. J. Med. 348, 255–256 (2003).

    Thomas CE et al. Progress and problems with the use of viral vectors for gene therapy. Nature Rev. Gen. 4,346-358 (2003)

    First CAR-T cell therapy trials:

    Kalos et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Science. 3(95):95ra73(2011)

    Maus MV et al. Adoptive immunotherapy for cancer or viruses. Annu Rev. Immunol. 32, 189-225 (2014)

    First use of lentivirus:

    Naldini L et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 272, 263-267 (1996)

    Penn Medicine news release, Carl June CAR-T cell.

    https://www.pennmedicine.org/news/news-releases/2015/march/ken-burns-cancer-documentary-t

    Joe Quinn, Temple University, Bioprocessing class

    - https://www.labome.com/method/Nucleic-Acid-Delivery-Lentiviral-and-Retroviral-Vectors.html

    - https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/

    - https://viralzone.expasy.org/264?outline=all_by_species

    Inotechopen, Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus:

    - https://www.intechopen.com/books/viral-gene-therapy/production-of-retroviral-and-lentiviral-gene-therapy-vectors-challenges-in-the-manufacturing-of-lipi

    Bioprocess International

    http://www.bioprocessintl.com/2016/emerging-platform-bioprocesses-for-viral-vectors-and-gene-therapies/

    - https://en.wikipedia.org/wiki/Lentivirus

    Cell culture dish
    http://cellculturedish.com/2015/01/2014s-top-ten-cell-culture-dish-ask-expert-sessions/
    Wave bioreactor
    https://www.bioprocessonline.com/doc/single-use-vs-stainless-an-economic-microbial-fermentation-comparison-0001
    T-flask
    http://www.cosmobrand.com/product/cell-culture/flasks/47_c871e3e421e64facb52f0660dbdea168.shtml
    Multi-tray
    http://www.techmate.co.uk/cell-factory-easyfill-2-trays-pk-6
    Spinner
    https://www.fishersci.com/shop/products/wheaton-magna-flex-spinner-flasks-8/p-123112
    Roller Bottle
    https://www.bioprocessonline.com/doc/cell-culture-roller-flask-system-cellroll-0001
    Bioreactor
    https://www.axetris.com/en/mfd/analytical-instruments/bioreactor-and-fermentor
    Large bioreactor
    https://www.genengnews.com/gen-articles/filterful-of-fiber-fiberful-of-hollow/5467?page=2

    ICH Quality Guildelines
    http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html