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AAV Gene Therapy

AAV Biology

Overview of gene therapy market

The first set of promising gene therapies were brought to a halt in 1999 after the death of Jesse Gelsinger from an immune reaction to the vector transporting a gene therapy for his metabolic disorder, and the development of leukemia by multiple patients—including one who died—in trials that ran between 1999 and 2002 for X-linked severe combined immunodeficiency (SCID-X). In the years since, better clinical and scientific understanding of the safety risks have enabled the first wave of clinical success. This has included a better understanding of immunogenicity and integration patterns of viral vectors as well as improved technologies and modified delivery mechanisms. Manufacturing improvements have included new chemistry, manufacturing, and controls regulations and improved accuracy of oligo synthesis.

More than 150 investigational new drugs applications were filed for gene therapy in 2018 alone. With this in mind, we expect this market to grow significantly, with ten to 20 cell and gene therapy approvals per year over the next five years. This growth is set to come from a wide range of modalities, from ASOs and RNAi5 —Spinraza and Onpattro being the first two therapies approved using this modality to potentially curative modalities deploying AAV6 and lentivirus therapies, such as Luxturna and Zynteglo. CRISPR (clustered regularly interspaced short palindromic repeats) gene editing–based therapeutics present a long-term growth opportunity, generating significant excitement and investment in the technology (more than $600 million invested in CRISPR start-ups by 2017 and the first in human trials expected to kick off in 2019), however they are unlikely to have significant clinical impact before 2025.

AAV vectors

As of 2019, much of the focus in development has been on monogenic rare diseases; all currently approved therapeutics fall into this category. Rare diseases tend to have clear genomic targets as well as high unmet need in small patient populations. These patients have generally been underserved by other, more traditional, therapeutic modalities (including monoclonal antibodies)—making them ideal targets for gene therapies.


Furthermore, this focus on high unmet need in smaller, underserved populations has enabled faster approval by regulatory authorities than diseases that impact larger patient populations. Most gene therapies have come to market under an accelerated regulatory review pathway (for example, a regenerative medicine advanced therapy or breakthrough designation by the FDA), which expedites the approval process. The importance of this accelerated process was emphasized in a May 2018 speech by then–FDA Commissioner Scott Gottlieb: “These products are initially being aimed at devastating diseases, many of which are fatal and lack available therapy. In these settings, we’ve traditionally been willing to accept more uncertainty to facilitate timely access to promising therapies.” 

These accelerated pathways are shifting the paradigm of clinical trials by consolidating the Phase I, II, III process into Phase I, Phase II/III, and confirmatory Phase III trials after approval (similar to the trend in oncology research). The small patient populations also make it possible for companies to experiment with innovative trials designs (with regulatory involvement and approval), including single-arm and novel or surrogate endpoints. However, these trials may also require a different approach to decision-making within biopharma operations.

Although rare disease remains a focus in gene therapy, much of the early-stage gene therapy pipeline is in oncology. As of September 2019, roughly 25 percent of the overall gene therapy Phase I and II pipeline is oncology focused, including 17 Phase I RNA based and 8 Phase I DNA-based therapies. These oncology-directed therapies will compete with more traditional modalities (many of which will soon have biosimilar competition), and thus will need to demonstrate increased cost-effectiveness.

Much of the innovation and development in gene therapy have been driven by smaller biotech companies or research universities, sometimes in partnership with a large pharma company or an entity specialized in the targeted therapy. In fact, 90 percent of gene therapy development to date is from companies with fewer than 500 employees. Many of these biotechs are platform companies who have optimized the manufacturing and delivery of their technology. When combined with the current funding climate, this has enabled many of them to quickly scale to multiple clinical programs across multiple therapeutic areas. As the technology underlying gene therapy matures, large pharma companies are becoming more excited about owning the technology versus partnering, as shown by the recent large acquisitions of Avexis by Novartis (for $8.7 billion) and Spark by Roche (currently in negotiation for $4.3 billion).

AAV Based Drugs

Glybera - Alipogene tiparvovec

Glybera (Alipogene tiparvovec) is a gene therapy treatment designed to reverse lipoprotein lipase deficiency (LPLD), a rare inherited disorder which can cause severe pancreatitis. Glybera is composed of an AAV1 viral vector with an intact copy of the human lipoprotein lipase (LPL) gene for delivery to muscle cells. Data from the clinical trials indicates that fat concentrations in blood were reduced between 3 and 12 weeks after injection, in nearly all patients. In July 2012, the European Medicines Agency (EMA) recommended it for approval (the first recommendation for a gene therapy treatment in either Europe or the United States), and the recommendation was endorsed by the European Commission in November 2012. Glybera was developed over a period of decades by researchers at the University of British Columbia and later Amsterdam Molecular Therapeutics (AMT), which acquired rights to with the aim of releasing the drug in Europe. After spending millions of euros on Glybera's approval, AMT went bankrupt and its assets were acquired by uniQure.


The vector genome contains the transgene expression cassette containing the cytomegalovirus (CMV) immediate early promoter, the cDNA sequence of human lipoprotein lipase variant S447X (LPLS447X), the bovine growth hormone polyadenylation site, and a woodchuck hepatitis virus post transcriptional regulatory element (WPRE) which is required to improve LPL gene expression. The applicant is using the WPRE of WHV in the vector genome without destroying the X-open reading frame present in this region. The expression cassette is flanked by two inverted terminal repeats (ITRs) derived from AAV2. The total length of the vector genome is 3.6 kb. The vector genome, either – or + strand, is pseudotyped with AAV serotype 1 capsids which are composed of 60 subunits formed by three viral proteins, VP1, VP2, and VP3, in a relative stoichiometry of 1:1:10. The major capsid protein is VP3, estimated to represent about 80% of the total mass.


Glybera was  the first gene therpay to be approved in Europe. However, Glybera gained infamy as the "million-dollar drug," causing its manufacturer, uniQure, to remove the drug after two years on the European market. As of 2018, only 31 people worldwide have ever been administered Glybera, and uniQure has no plans to sell the drug in the US or Canada.

Luxturna-Voretigene neparvovec 

Voretigene neparvovec (Luxturna) is a novel gene therapy for the treatment of Leber's congenital amaurosis. It was developed by Spark Therapeutics and Children's Hospital of Philadelphia. Luxturna uses the AAV2 to carry a functional copy of the RPE65 gene into the retinal pigment epithelial (RPE) cells to compensate for the RPE65 mutation. It is the first in vivo gene therapy approved by the FDA. Leber's congenital amaurosis, or biallelic RPE65-mediated inherited retinal disease, is an inherited disorder causing progressive blindness. Voretigene is the first treatment available for this condition. The gene therapy is not a cure for the condition, but substantially improves vision in those treated. It is given as an subretinal injection. 


The voretigene neparvovec genome contains the following components:

1) the cytomegalovirus (CMV) enhancer;

2) the chicken beta actin (CβA) promoter;

3) the CβA exon 1 and intron;

4) the cloned cDNA coding for human retinal pigment epithelium 65kDa protein (hRPE65)

5) the bovine growth hormone polyadenylation (PolyA) region,

6] Two  AAV2 ITRs.

7] Small intervening non-functional DNA sequences derived in the process of assembling the genetic elements through recombinant DNA techniques are also present.


Zolgensma-Onasemnogene abeparvovec

Onasemnogene abeparvovec, sold under the trade name Zolgensma, is a gene therapy medication used to treat spinal muscular atrophy (SMA). It is used with corticosteroids as a one-time injection into a vein. It was approved in the United States in 2019 for children less than two years old. Common side effects include vomiting and increased liver enzymes. Serious side effects may include liver problems, low platelets, and heart damage. Onasemnogene abeparvovec works by providing a new copy of the gene that makes the human SMN protein. 

Onasemnogene abeparvovec was developed by AveXis, which was acquired by Novartis, based on the work of Martine Barkats from the Institut de Myologie in France. It carries a list price of US$2.125 million per treatment, making it the most expensive medication in the world as of 2019.


The voretigene neparvovec genome contains the following components:

Zolgensma 3.jpg

Hemophila  Gene Therapy


Hemophilia A is a rare blood disorder caused by mutations in the F8 gene that provides instructions for making factor VIII, a protein that plays a crucial role in forming blood clots. It is the most common type of hemophilia and occurs much more frequently in males; incidence is estimated at 1 in 4,000-5,000 male births. The mutation causes factor VIII to be reduced in amount, absent, or dysfunctional. Because factor VIII is necessary for normal blood clotting, hemophilia A patients have delayed clotting and require regular infusions of either plasma-derived or recombinant factor VIII to prevent life-threatening bleeding episodes.

Haemophilia B is a blood clotting disorder causing easy bruising and bleeding due to an inherited mutation of the gene for factor IX, and resulting in a deficiency of factor IX. Hemophilia B is four times less common than hemophilia A.

At present, BioMarin Pharmaceuticals, Spark Therapeutics, Pfizer, and UniQure all have gene therapy products being evaluated in phase III studies. The BioMarin and Spark products are FVIII gene transfer products and the Pfizer and UniQure products target FIX (see TABLE). Each of these companies have reported factor activity levels returning to the normal range in patients with mild hemophilia in phase I and II trials, although not all these data have been published in peer-reviewed journals

Hemophilia A.png

Valrox (valoctocogene roxaparvovec), also known as BMN 270, is an investigational gene therapy being developed by BioMarin Pharmaceuticals for treating hemophilia A with a single-dose intravenous injection. Valrox contains a harmless virus called AAV5 that carries a healthy copy of the factor VIII gene under the control of a liver-specific promoter. Promoters are regions in the DNA that dictate where a certain gene will be active. When Valrox is infused into hemophilia A patients, the recombinant factor VIII gene is delivered into the nuclei of the cells of several tissues, including the liver. However, factor VIII protein is made only in the liver cells because of the liver-specific promoter. It then is secreted into the bloodstream.

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