When the Price is Right for Gene Therapy


A lot of hype and pomp has been thrown around about the cost of gene therapy treatments recently. The current costs are truly staggering, ranging from over $300,000 up to $1 million for a one-time treatment. The truth of the matter is that every new technology that comes on the market will be expensive at first due to supply and demand as well as the costs for initial development. With time and with technology to improve manufacturing the costs will change (hopefully) for the better. When the automobile came on the market in 1900 the average cost was over $1000 while the average family earned $750 a year. Over the next 25 years prices declined by 11% per year until in 1924, the Ford Model-T car cost just $265.

Cars through the ages:

By the end of the century the price of a family car had fallen by 50%, when adjusted to inflation. In just a few dozen years, with the added enhancements in safety and technology, the drop in price meant that every family could have their own car. This vast reduction in cost was primarily because cars could be made on an assembly line and could be sold in a production cycle. In the same way, I am betting that costs of many gene therapy treatments will decline greatly over the next century, due to improved technologies and an economy of scale. This week I want to dissect some factors that determine how gene therapy is really priced.

Firstly, the bad news: AAV and Lentiviral gene therapies currently have astronomical costs. Here are 5 most expensive gene therapy drugs that have been approved today and their eligible patient populations:


Glybera has already been discontinued due to a lack of market demand - only one person has ever been treated. Three other gene therapies have also been withdrawn in Europe since approval by the EMA due to unsustainable costs and tighter controls on drug pricing. In fact, all of the approved therapies currently face market difficulties because they treat rare or ultra-rare diseases.

A rare disorder in the US, termed “Orphan disease” affects less than 200,000 people (620 patients per million) while an Ultra-Orphan disease can affect as little as 1 in 50,000 people (20 patients per million). Since there are so few patients, companies want to charge a higher price for their gene therapy. However, if we only allowed the free market to dictate drug prices all of these gene therapy treatments will have to be shelved. Yescarta, the treatment for B-cell lymphoma in adults, manufactured by Kite Pharma (now Gilead), could treat up to 7,500 people per year. This remains the largest treatable population so far by any marketed gene therapy product. Furthermore, the United States, unlike its European counterparts has traditionally allowed the free market to determine drug prices and this has led to short-term monopolies that jack up drug prices. In some ways this has been due to limited patent life and legislative blindspots (Just look at Gleevec).

However, drug prices for gene therapies are not determined by supply and demand alone. The costs for society and for the patient factor in a great deal. An article in Science, in 2016, written by Stuart Orkin and Philip Reilly provides a great summary about this issue. Cystic fibrosis, as an example costs the average patient $25,000 per year in terms of general health support and lifetime costs range around $750,000. Hemophilia A, another more common genetic disease currently targeted for gene therapy trials, costs patients around $300,000 per year and up to $10 million over a lifetime. In fact, even though each individual Orphan disease affects a few people, aggregating all the rare diseases reflects the huge burden that the health care system faces today. If we were to add up current treatments for sickle cell disease for 70,000 patients in the US they would exceed $1 billion per year, not to mention the days lost from work and family burden. Thus, the high economic costs of rare diseases for society go some way towards justifying the high costs of gene therapy.

Orkin and Reilly, Science, 2016, current costs for managing healthcare in genetic diseases:



The Price of Development and Production
Several factors are involved in the determining the price for development and production of gene therapies.

Since genetic disorder and the standard of care varies drastically between disease states, the procedures for each specific gene therapy will be different. For example, procedures for treating hemoglobin disorders and neurodegenerative diseases may involve extraction of patient hematopoietic stem cells, ex-vivo treatment with a lentiviral vector, treatment of the patient with immunosuppressive drugs and then readministration of the modified cells. On the other hand, treatment of a patient with retinal dystrophy may involve simply injection of AAV directly into the eyes. If multiple administrations are needed to maintain gene expression and quality of treatment the costs will naturally ascend over a patient’s lifetime. Therefore the mode of treatment directly affects the cost of the therapy.

The costs of manufacturing different viral vectors will also vary widely depending on the target organ for treatment, quality control of viral vectors and the process by which viruses are synthesized at scale. For example, treatment of muscular dystrophy involves injection of virus into multiple muscle group whereas treatment for blindness involves injection just into the eye. Significantly higher vector quantities would be needed for the former treatment over the latter.

Each gene therapy takes many decades to develop and possibly some failed clinical trials. In addition pharmaceutical and biotech companies often have several candidate treatments in the pipeline which may benefit more patients in future compared to its currently market-approved therapy. Furthermore, the costs for submitting an IND and BLA for FDA approval are substantial. The biotech industry has already invested over $10 billion in gene therapy over the last 20 years without taking back any revenue. At some stage biotech companies must decide on how to make up for sunk costs. This may well be through charging more for the currently approved product.

For ultra-rare disorders such as Tay-Sachs disease affecting less than 1 in 250,000 people, market incentives alone do not encourage drug development. Joint efforts between the FDA, National Institutes of Health and the biotech/pharmaceutical companies will have to be initiated to come up with ways to treat such diseases.

Finally, the most crucial factor that determines the price of gene therapies is the outcome of patients. For example, a child who is treated for bone marrow cancer and is able to live a meaningful life but with slightly compromised health will have a favored treatment compared to a child who is given only a few years left to live. That treatment must be significantly better than current therapies in order for it to be brought to approval. A good disease outcome may play a large part in charging high annuity payments, with an initial large lump sum followed by smaller payments later.

New Models for Paying the Cost

Solutions for paying for these large costs could come in a number of ways:

- One way would be to pay for expensive gene therapies in a large up-front payment and have the drugmaker or its successor bear the burden of of the remaining reimbursements. Some companies, like Vertex, are already shifting towards this model. When Kalydeco was approved as a small molecule treatment for Cystic Fibrosis, its cost was $300,000 per year per patient. A look at the cystic fibrosis patient communities showed that when a person’s health insurance coverage is depleted, Vertex comes in to fill the gap. One patient paid $5000 up front for the first fill and then subsequently paid $50 as co-pay and $15 out of pocket per month. The Cystic Fibrosis Foundation dub the model “venture philanthropy” in which the foundation itself originally paid $3.3 billion to the then Aurora biotech company (which became Vertex) to develop Kalydeco.

- Another way would be to find new regulatory processes at the FDA that would streamline the approval processes for ultra-rare disorders. Drug makers already rely on the Orphan Drug Act to cut costs since the act reimburses medical companies for developing treatments for rare disorders. This would reduce the pricing of copays. This is already being done since clinical trials for Orphan-designated diseases, such as spinal muscular atrophy and muscular dystrophy are shorter and less expensive. The FDA also has a variety of expedited pathways set up for breakthrough therapies that can show significant benefit for patients.

- A third way would be for medicare to provide reimbursements. This week it was revealed that the government healthcare program will pay the $395,380 cost to hospitals for Yescarta as an outpatient treatment. However, patient hospital costs are currently capped at a deductible of $1340. Furthermore, Medicare will only pay up if Yescarta can be shown to significantly improve health outcomes of patients within the first month.

However we pay for gene therapy in future, one thing is for certain, society must come to an agreement with the biotech industry about costs. This will boil down to a balance between funding drug companies to developing cures for more common disorders and enabling patients and caregivers to get access for desperately needed treatments. If Henry Ford could find a way to reduce the cost of an automobile to an affordable level in just 25 years then we in the medical and scientific community must surely find a way to reduce the cost of gene therapy treatments which, unlike a car, will be more vital for a patient’s survival.



Reilly P, Orkin SH, Paying for future success in gene therapy, Science, 2016: 352(6298) 1059-1061.






EU approved and withdrawn gene therapies


Gleevec, Cost Increase


Plos blog, Kaledyco payment model


Medicare Reimbursement


Bureau of Labor Statistics


The Economist, Price changes over the 20th C








    Lentiviruses - Not just Retro Chique


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):


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):


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):


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:


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.




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.


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


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

Cell culture dish
Wave bioreactor
Roller Bottle
Large bioreactor

ICH Quality Guildelines


    Luxturna / Voretigene - FDA's first AAV Approval



When FDA first announced its approval of Luxturna (voretigene neparvovec-rzyl), sponsored by Spark Therapeutic, Inc in December 2017 the FDA Commissioner, Scott Gottleib made a press release about how gene therapy has become a breakthrough in the treatment of rare, intractable illnesses. It was also one of three gene therapy approvals in one incredible year for the agency, which has traditionally taken a cautious stance on novel, large molecule (and therefore) complex therapies. However, on close examination of Luxturna’s now public documents and advisory committee meetings, it seems the drug development process was given due diligence.

Luxturna is a recombinant adeno-associated virus of serotype 2 (rAAV2) expressing the gene for human retinal pigment epithelial protein 65 kDa (hRPE65). It is indicated for the treatment of patients with bialleic RPE65 mutation-associated retinal dystrophy. The mutations occur in only 1000-2000 patients in the US who belong to a wider group of people suffering from genetic retinal disorders and includes the disease Leber congenital amaurosis (LCA). The human RPE65 protein is an essential protein for converting 11-cis-retinal to all-trans-retinal in the visual cycle that is critical for maintaining vision:

Travis 2007

People with these mutations mostly get affected early in life. They find themselves with severe vision impairments which progress to night blindness and loss of visual field.

The AAV plasmid is designed with a CMV enhancer and a beta-actin promoter to drive robust expression, based on original research by Jean Bennet at UPenn. The pre-clinical and IND-enabling studies were done in mice and dogs modeled genetically on the human RPE65 mutation. The dogs in particular showed significant improvement after viral vector injection into the eye over a two and a half year period, provided they were dosed early in life.

Luxturna was approved following a Phase 1 study and a Phase 3 study. The IND was submitted in 2007 by Children’s Hospital of Philadelphia and granted an Orphan Drug designation in the following year. The IND was then transferred to Spark Therapeutics, Inc in 2014 which became the sponsor up to its approval. In 2014 the FDA also granted Breakthrough Therapy Designation, which further expedited the development process.

A brief summary of the clinical trials:

Phase 1:

The Phase 1 study was an open-label dose-exploration safety study on 12 patients who were first given the gene injected into one eye to observe any toxic effects or adverse events. Preliminary efficacy assessments were also obtained for visual function. This was followed by injection into the second eye a few years later.

The inclusion criteria included patients who were 8 years or older, with visual acuity of no better than 20/160 (20/20 vision being perfect vision).

The exclusion criteria included:
Insufficient viable retinal cells
Neutralizing antibodies to AAV at greater than 1:1000
Pre-existing eye conditions that would interfere with surgery
Ocular surgery within previous 6 months.

Three dosing plans were set for the first eye: 1.5 X 10^10 vg/ 150uL, 4.8 X 10^10 vg/150uL and 1.5 X 10^11 vg/300 uL. For the second eye they used the highest dose since no adverse events were present in the first eye. To minimize immune response oral prednisone was given around the time of administration.

Phase 3:

The Phase 3 study was an open-label randomized study involving 31 patients. Again the gene was injected into one eye first before it was injected into the second eye - this time waiting for just a week or two. After one year the control patients and test group patients were crossed-over. Clinical assessments were done for safety and efficacy. A long-term follow up plan was designed for 15 years.

Subretinal injection procedure:

Phase 3 trial design and follow-up scheme:

The efficacy test involved using the multi-luminescence mobility testing (MLMT) assessment in which patients were asked to navigate a course laid out be a number of arrows, turns and obstacles and at varied light illumination conditions. Patients were assessed at baseline, Day 30, 90, 180 and 365 using one treated eye, then the other treated eye. Every run of the test was recorded with video tape and assessed. Scores were given based on how well a person could complete the course in low light (high score for passing) or bright light (lower score for passing). A score change of 2 from before to after treatment was considered clinically meaningful.

The inclusion and exclusion criteria for Phase 3 trials were much the same as Phase 1 except subjects were 3 years or older and they had to be able to perform MLMT testing.

Dosing was set at 1.5 X 10^11 vg/300 uL injected subretinal.

From the efficacy study it was found that patients with the treatment had an increase in MLMT score which was significantly more than in the untreated eye after one year. This was found in 51% of patients with treatment in both eyes and 71% of patients with treatment in just one eye after one year. Interestingly no statistical significance was found in patient visual acuity improvement but there was for sensitivity to light (light sensitivity threshold).

The major adverse events discovered from the Phase 3 trial included Conjunctival hyperemia, intraocular pressure, cataract development and retinal tear. Two of the patients suffered serious adverse events with permanent visual loss.

For each study safety assessments were done. They carefully looked at Interferon-gamma responses to AAV2 and to RPE65 by ELISPOT assay in peripheral blood mononuclear cells (PBMCs). For most measurements across the Phase 1 and 3 studies there was minimal, low or no change in antibody titers to AAV capsid and RPE65.


HEK293 cells were triple transfected with rep/cap, helper and the RPE65 plasmids before the virus was harvested. The harvested virus was then purified through the downstream process. Using this manufacturing process they were able to produce up to 3e15 vector genomes necessary for Phase 1 trial and make them within GMP limits for quality. A detailed look at the upstream and downstream process can be found by in a paper published by James Fraser Wright in 2010:

AAV Manufacturing Process Flow Through:


Manufacturing Facility Diagram:

Although retinal dystrophies are a complex disease characterized by both dysfunction and degeneration of photoreceptors, the Luxturna AAV seems to have overcome this problem by providing restored function in patients with the RPE65 mutation. It remains to be seen whether this treatment has long last benefits which stretch out beyond the 1-2 year period of analysis seen in patients so far, onto adult and old age when retinal degeneration becomes more common place.



- Expert analysis of plasmid CHOP, Spark and UCL:


- Advisory Committee Meeting:


- Luxturna FDA Supporting Documents:


- Original Research:

Wright, J. F., Wellman, J. & High, K. A. Manufacturing and regulatory strategies for clinical AAV2 hRPE65. Curr Gene Ther 10, 341-349 (2010)

Acland, G. M. et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28, 92-95, doi:10.1038/88327 (2001)

Amado, D. et al. Safety and efficacy of subretinal readministration of a viral vector in large animals to treat congenital blindness. Sci Transl Med 2, 21ra16, doi:10.1126/scitranslmed.3000659 (2010).

Travis GH, Golczak M, Moise AR, and Palczewski K. Diseases Caused by Defects in the Visual Cycle: Retinoids as Potential Therapeutic Agents. Annu. Rev. Pharmacol. Toxicol. 2007; 47:469–512



    AAV - An Awesome Vehicle!



As I promised last week, this will be the first in a series of blogs on gene therapy in which I will introduce what viral vectors are and how they have become a definitive weapon in the battle to cure genetic diseases. This week I will focus on AAV.

Since its discovery in 1965, the Adeno-Associated Virus (AAV) has come a long way in its usefulness as a molecular tool. Originally found by Bob Atchison, M. David Hoggan and Wallace Rowe, AAV was known to be a parvovirus made of capsids containing single stranded DNA and inverted terminal repeats (ITRs). The ITRs are self-priming hairpins that enable genome replication. The capsid proteins encoded by the viral genome, VP1, VP2 and VP3 give the virus its characteristic icosahedral shape, its ~25 nm diameter size and its viral properties. AAV binds to cell surface receptors in order to enter the host cell, rather like any other virus. The most common receptors targeted by AAV include the Heparin Sulfate Proteoglycan receptors (HSPG) and integrin receptors.

AAV structure:

Left Panel: Computer simulated AAV model at 22 nm in diameter. Middle Panel: Electron Microscopy visualization of AAV particles. Right Panel: AAV rep / cap genes and the encoded VP1 / VP2 / VP3 proteins. Pictures courtesy of SignaGen Laboratories.

AAV, pic

AAV entrance into the cell - typically AAV binds to HSPG receptors, enters by endocytosis at the membrane, gets carried in the endosomes and escapes through into the nucleus to regulate gene expression:


When the virus takes over cellular machinery, more than 100,000 particles can be produced by one cell. Moreover, AAV is not a pathogen. It can persist in the host cell for months without triggering any adverse symptoms. AAV can therefore be studied safely in Biosafety Level-2 (BSL-2) laboratories, with minimum sterilization, clean room specifications and decontamination procedures.

AAV vectors are currently the most commonly used gene therapy based vectors. The first clinical trial to test recombinant AAV (rAAV) gene therapy happened in 1993, to test a treatment for cystic fibrosis, attempting to replace the monogenic mutation of CFTR protein with a healthy one. Despite early setbacks in the clinic the technology has now improved leading to better safety and efficacy. Today there are hundreds of rAAV based therapeutic products being developed. In 2012 the EMA in Europe approved Glybera, a rAAV-1 based gene therapy for familial lipoprotein lipase deficiency (LPLD), manufactured by the dutch company UniQure. Last year, the FDA approved Spark Therapeutics’s Luxturna, the rAAV2 based gene therapy for certain forms of retinal dystrophy, including Leber Congenital Amaurosis.

rAAV gene therapy products (picture courtesy of Drug Development and Delivery website:


Some of the main advantages of the rAAV vector system include ease of producing large yields of virus, long-term robust transgene expression, its tropism for non-dividing and dividing cells and lack of adverse effects associated with its administration. Different rAAV vector serotypes occur naturally and target different cell receptors giving them specificity for target tissues. With the advent of directed evolution techniques, scientists have engineered hybrid and synthetically derived rAAV serotypes that have even higher target specificity for diseased tissues and can evade pre-existing immunity. This has greatly expanded the commercial potential for using rAAV-based gene therapy.

AAV Serotypes:

AAV Serotypes

Making the vehicle:

The traditional procedure for making rAAV in the lab at small scale starts off by transfecting mammalian cells (usually HEK 293 cells) with three plasmids:
Ad Helper plasmid - genes that help the virus to replicate
Rep/Cap plasmid - the regulatory genes and structural genes that package the virus
Transgene plasmid flanked by the ITRs - encode the therapeutic gene.

When the cells start expressing these genes, rAAV particles are made. To harvest these particles, cells are lysed and the lysate is ultra-centrifuged through cesium chloride gradients in order to concentrate the virus. The purest virus fraction is collected from the gradient, dialyzed in PBS and stored for use.

Traditional rAAV production in the lab (Picture courtesy of SignaGen Laboratories):

AAV making


Now that clinical grade rAAV is in demand, increasingly Good Manufacturing Practices (GMP) production solutions are needed. GMPs are founded on Joseph Juran’s trilogy of principles, Quality Planning, Quality Control and Quality Improvement. A Quality by Design approach must be taken by manufacturers in order to build quality into the process as well as the product. This is taken seriously by the FDA, EMA and other global regulatory authorities when reviewing drugs for approval. GMP guidelines for pharmaceutical development, quality risk management, quality systems and development of drug substances are enshrined in the International Conference of Harmonization documents, named ICH Q8, Q9, Q10 and Q11. These documents require manufacturers to come up with Quality target product profile (qTPP) and Critical Quality Attributes (CQAs) that must be set up to determine key properties of the drug are within acceptable, safe limits at all times during the manufacturing process.

What does this GMP jargon have to do with rAAV therapy? To produce rAAV for clinical use there are two general processes: the Upstream Process and the Downstream Process. The upstream process is geared towards transfecting cells with the viral plasmids, seeding cells, growing up the cells in a bioreactor and harvesting the virus. It is important for the Master Cell Bank to be characterized, quality checked and stored with meticulous notes since this is the source from which you will will derive the Working Cell Bank, which will be seeded. The downstream process aims to purify the harvested bulk through a series of purification and chromatography columns before retaining the virus, formulating it and freezing or lyophilizing it in a bottle. A combination of cation exchange, anion exchange and tangential flow filtration techniques are often employed for the purification process.

Upstream Process:

Downstream Process:

In order to define CQAs a manufacturer must make challenging decisions on a variety of parameters during the production process. Chief among these questions involve:
- What type of cell line do you use in order to make the virus? Mammalian cell line or insect cell line, such as baculovirus?
- Do you transiently transfect the cell lines once they have been seeded from the cell bank? Or do you use stable cell lines that have been frozen with the Helper plasmid, Rep/Cap plasmid and Transgene plasmids? Do you use a helper adenovirus to infect the cells with the desired viral plasmids or do you transfect by traditional calcium phosphate or PEI method?
- What type of cell growth medium and container do you use? Do you start with adherent cells in stacked flasks and build up to larger and larger bioreactor tanks or do you drop cells into a bioreactor to start with?
- Do you choose single-use bioreactors and disposable culture systems or do you re-use culture vessels that require cleaning after each use?
- What types of purification systems do you use? Cation Exchange Chromatography or Anion Exchange Chromatography?

Linking manufacturing challenges to regulatory requirements is tricky. Once rAAV vector production begins it will be essential to test batch-to-batch variations for quality. This involves testing for identity, purity, stability and potency by sampling at different stages along the production cycle. Impurities are a great challenge as well. Between 1% and 8% of purified rAAV particles are known to contain incorrect nucleic acid sequences. That can be a huge number of incorrect particles in a single batch of virus.

Along with the lack of knowledge base about rAAV (since the AAV structure is so complex relative to small molecule drugs), the other disadvantage of rAAV manufacturing is the impurity profile. There are Process-related impurities and Product-related impurities. Process-related impurities are derived from the manufacturing process. Raw components, cell substrate, cell culture medium and residual helper components could all contribute to these impurities. Product-related impurities result from the vector itself which interfere with efficacy and safety. These can include empty AAV capsids, non-infectious rAAV particles, aggregated, oxidized and degraded vectors, nucleic acid impurities, minor variations in stoichiometry of VP1, 2 and 3.

Process-Related Impurities:

Product-Related Impurities:

Both genotoxicity (potential for causing cancer) and immunotoxicity (potential for causing an adverse immune reaction) are potential threats to the production of AAV. Therefore, as a Quality Control step, a manufacturer must set up a Risk Assessment plan for each and every one of the impurities in the tables above.

As mentioned earlier, a single cell can generate many thousands of rAAV particles. However, in order to be useful in the clinic, rAAV-based viral vectors must be produced in even larger numbers, between 10e11 to 10e14 genomic particles (vector genomes / vg). Current GMP manufacturing scales for rAAV often fall short of this. Part of the reason is that smaller quantities of rAAV are satisfactory for the smaller scale clinical studies on rare “Ultra Orphan” diseases which only impact a few hundred individuals per hundred million people. However, as more common diseases are studied for rAAV therapy and as clinical trial phases advance from proof of concept to toxicity/Phase 1 studies onto Phase 2 / 3, more virus will need to be generated. This means scaling up from adherent cells to large bioreactors, providing a clean room environment within class 10,000 and having more stringent quality control parameters to ensure quality in production.

Genomic particles vs Phase of Clinical Trial, a simplified graph of how many genomic particles are required for each phase of rAAV development:


Progress of cGMP compliance at different stages of product development

A vision for the future

There are more than 7000 rare diseases that remain untreated. The use of rAAV as gene therapy is just one of a growing arsenal of weapons scientists are currently developing to fight these disorders. There are limitations to using rAAV, such as its packing size (usually no more than 5000 bp transgene can fit), the problem of neutralizing antibodies that stop the action of beneficial rAAV particles and problems with endosome escape once rAAV is inside the cell. Selecting novel rAAV capsids by directed evolution would be one way to overcome some of these deficiencies. However, it is possible that if rAAV is to be used in the future it will have to be done in combination with other drugs that mitigate these deficiencies.

Next week I will do a mini-case study into FDA's newly approved Luxturna for the treatment of RPE65 mutation-associated retinal dystrophy. The therapy that has enabled visually impaired patients to see clearly for the first time.


AAV pictures from:



Production of AAV: Chandler RJ, Venditti CP, Gene therapy for metabolic diseases, Transl Sci Rare Dis. 2016;1(1):73-89.

Daniel C. Smith, AAV Vector manufacturing - Challenges and Opportunities in the Manufacturing of AAV Vectors Used in Delivery of Gene Therapy Treatments, Drug Development and Delivery, 2/28/2017

A-Mab Case Study: http://c.ymcdn.com/sites/www.casss.org/resource/resmgr/imported/A-Mab_Case_Study_Version_2-1.pdf, — upstream/downstream

Impurities picture: Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment. J. Fraser Wright. Biomedicines, 2014:2, 80-97, — Impurities

Hasti E, Samulski RJ, Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective. 2015, 26:257-265

GMP Compliance picture: Van Der Loo JCM, Wright JF, Progress and challenges in viral vector manufacturing. 2016: 25(1)42-52. — cGMP compliance phases figure

Keeler AM, Flotte TR, Gene Therapy 2017: Progress and Future Directions. 2017; 10:242-248



      The Year of the Gene Therapy


    Gene Therapy

    As the lunar new year dawned last week I thought about the latest scientific accomplishments that have been achieved in my field and what they mean for me as a life scientist.  Now that I am working on developing gene therapy treatments I find myself with a front row seat to possibly one of the biggest breakthroughs in human history – the eradication of genetic diseases at their source.

    In many ways 2017 was a breakthrough year for gene therapy. The FDA approved several new treatments designed to cure devastating genetic diseases.  These included Kymriah, a CAR-T cell therapy indicated for childhood B-cell lymphoblastic leukemia; Yescarta, a T cell immunotherapy for the treatment of B-cell lymphoma in adults; Imlygic, an oncolytic viral therapy that treats melanoma and Luxturna, an AAV gene therapy that corrects the mutation causing retinal dystrophy. Other genetic diseases such as sickle-cell disease and hemophilia are also seeing clinical trials come to fruition both in the US and across the world.  With the flood gates open for such treatments, this year the FDA is likely to approve even more gene therapy products.

    Gene therapy requires the use of viral vectors (adeno-associated viruses (AAV), adenoviruses, lentiviruses and gamma-retroviruses), or cationic liposomes, to deliver healthy genes to patients suffering from inherited diseases caused by mutated genes.  The idea of replacing faulty genes with functional ones in patients has been around for decades and twenty years ago it was all the rage.  However, the death of Jesse Gelsinger in 1999 while on a clinical trial for his X-linked genetic disease, put a halt to the field. It was not until a few years ago when there was a resurgence in interest for using viral vectors.  In the intervening years, many scientists have labored in the shadows improving viral vectors for better safety and efficacy, while overriding the body’s natural immune response to neutralize or (worst case scenario) to react against viruses.

    A brief history of gene therapy (Keeler et al., 2017):


    It is on the back of these efforts that I am able to come in today to test new viral vectors for treating diseases like cystic fibrosis.  It could take years before my current research gets approval for treatment.  In the meantime there is much work to be done – gene therapy clinical trials are now going on everywhere and there is a race between various biotech companies to get the first approval in for each disease treatment.  Here’s to hoping and expecting that 2018 brings in some extraordinary treatments.

    To help people understand this field, I want to dedicate my next few blogs to a more in-depth review of AAV as a gene delivery product and do a case study on the Luxturna treatment for blindness.







    Keeler et al., Clin. Transl Sci, 2017, 10(4): 242-248: Gene Therapy 2017: Progress and Future Directions





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Previous Posts

Theranos - Moral Lessons for the Biotech Industry

Gene Therapy for Spinal Cord Injury

FDA - Golden Age for Gene Therapy

Pricing Gene Therapy

Lentivirus - Not just Retro Chique


AAV - An Awesome Vehicle!

The Year of the Gene Therapy

Masters in QARA

Industry High Castle


Regeneration Paper Out

Oligonucleotide Therapeutics

Brain-Spine Neural Interface

Black Mirror

A Journey into my Genome (2): Volunteering my DNA

ImPACT Traumatic Brain Injury

Retiring the Mouse Model Gold Standard

Brexit Britain I weep for you


Seven Years in Visaland

Photo Website

Restimulating the Party

Start Talking Science

A Journey into my Genome

Patent Law IX, The Limits of Biotech Patents

Patent Law IX, The Longest Patent Extension Battle

Patent Law VIII, Invasion of Patent Trolls into Biotech

Patent Law VII, DTC Genomic Testing

Patent Law VI, Supreme Court and Laws of Nature

Patent Law V, The Dark Web

Patent Law IV, Gaming the Hatch-Waxman Act

Patent Law III, The Brave New World of Biosimilars

Patent Law II, The Everlasting Patent

Patent Law I, CRISPR-Cas9

FDA Law Intro

The Big Idea

Accountability for Retractions

Neuroscience Drugs

Locked-in Syndrome

SCI scar Inhibitor




Neuropathic Pruritus

Mitochondrial Disease, 3 parent baby

Multiple Sclerosis and Axon Injury

Pint of Science Philadelphia

The Mesoscale Connectome

Tracing Neuronal Circuits

Pint of Science


The Brain Initiative

Two more online courses done

Fellowship Awarded

One week

Shriners Fellowship

PVA Fellowship

SfN Itinerary

Online Course Certificates

Systems Biology