Ups and Downs of Adenovirus and Adeno-Associated Virus Vectors in Gene Therapy

by Liana Relina Contributor , Ilia Petrov    

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Topics: Novel Therapeutics   

Gene therapy is still an investigational technique. Viruses are commonly used as vectors to deliver a needed gene to a host (defective) cell. An ideal viral vector is supposed to be genetically stable, non-toxic, and non-immunogenic, to have a high packaging capacity along with other important features. 

There are several types of viral vectors. Adenoviruses seemed to be promising ones. Their genetic information is encoded in double-stranded DNA. To engineer adenovirus vectors, the early genes (responsible for the replication of the viral genome and expression of the late genes encoding structural proteins of the capsid) are removed and replaced with a transgene. Adenovirus vectors can hold inserts as long as ~36 kb.  The adenovirus DNA is not incorporated into the host DNA. This peculiarity prevents insertional mutagenesis (an undesirable phenomenon observed in gene retrovirus-using therapy, as retroviruses have a specific enzyme, integrase, that can insert the genetic material of the retrovirus into any site in the host genome, sometimes resulting in malignancies; that was the reason of termination of clinical trials when leukemia resulting from insertional mutagenesis was reported in patients who received such gene therapy).  

The DNA molecule of an adenovirus vector remains extrachromosomal in the host cell nucleus, but it can be transcribed like any other gene. The only difference is that this transgene is not replicated when the cell divides so the descendants of that cell will not have the transgene. Therefore, adenovirus vector-based therapy requires re-administration as the cell population grows. 

The strong immunogenicity of adenoviruses prevents them from being ideal vectors. Most adult patients have been exposed to adenoviruses, as the latter are common pathogens in humans. Hence, the immune system will attack an adenovirus therapeutic vector just like any other adenovirus. The immune system will recognize proteins (antigens) on the adenovirus vector capsid and destruct them before the therapeutic transgene is delivered to the nucleus. Several strategies are employed to “deceive” pre-developed immunity against adenoviruses, such as using non-human adenovirus vectors (e.g., chimpanzee-derived), shielding the adenovirus surface with polymers, or encapsulating vectors into microspheres. Despite the advances in adenovirus vector engineering, they can still trigger the immune response.

In early studies, a number of attempts were made to deliver normal genes to compensate for dysfunctional or deficient ones, which were responsible for several human genetic diseases. Cystic fibrosis is a genetic disease attributed to a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). An adenoviral vector was used to deliver “healthy” CFTR genes to lung tissues. An adenoviral vector was also used to deliver the gene encoding ornithine transcarbamylase, which is a key enzyme in the urea cycle and is responsible for ornithine-transcarbamylase deficiency (X-linked genetic disease of the liver). 

These studies faced several challenges, including immunity to adenoviral vectors upon repeated administration of the vector, cellular cytotoxicity, and even oncogenesis. The most serious concerns about the safety of adenovirus vectors were raised in 1999 after Jesse Gelsinger died while participating in a gene therapy trial. Gelsinger suffered from ornithine transcarbamylase deficiency. His liver was unable to metabolize ammonia – a byproduct of protein cleavage. Gelsinger was enrolled in a clinical trial managed by the University of Pennsylvania. He was injected with an adenoviral vector carrying a normal gene. Gelsinger presented with a massive immune response triggered by the viral vector, which led to multiple organ failure and brain death. After his death, all gene therapy trials in the US were suspended. The Gelsinger case was a severe setback. Since then, work using adenovirus vectors has been focused on genetically crippled versions of viruses. The adenoviral immunogenicity and cytotoxicity were suspected to be due to the expression of several viral proteins. The newer generations of adenoviral vectors had these adenoviral genes removed. Nowadays it is even possible to engineer adenovirus vectors by removing almost all viral genes. These are so-called “gutless” vectors. Different adenoviruses vectors were tested to treat parotid salivary dysfunction, varicose ulcer, macular degeneration, angina pectoris/myocardial infarction, pancreatic carcinoma, neuroendocrine carcinoma, breast carcinoma, glioma. 

Adeno-associated virus (AAV) consists of a capsid, which is composed of three viral proteins encapsulating a 5kb single-stranded DNA. There are about a dozen naturally occurring AAV serotypes. AAV vectors show low immunogenicity. The paramount limitation of AAV vectors is the small size of a therapeutic gene insert. The maximum insert is about 4.7 kb long, which makes targeting larger genes challenging. AAV vectors were tested to treat the following diseases: Rett syndrome, cystic fibrosis, Huntington’s disease, hemophilia, Duchenne muscular dystrophy. The advantages and disadvantages of adenovirus and AAV vectors are summarized in Table 1.

 

Advantages 

Limitations

Adenovirus vectors

High DNA packaging capacity (8-38 kb)

High immunogenicity

High transduction efficiency

Transient gene expression 

Infect a wide range of cells and tissues,

both dividing and non-dividing cells                 

Pre-existing immunity

High expression level

 

Non-integrating

 

AAV vectors

Low immunogenicity

Low DNA packaging capacity (~4.7 kb)

Non-pathogenic

Production is technically demanding

Selective transduction

 

Non-integrating 

 

(Adopted from https://biovian.com/viral-vector-and-gene-therapy-basics-summarized/)

The first gene therapy product to be approved to treat cancer was Gendicine manufactured by Shenzhen SiBiono GeneTech Co.,Ltd. It is an engineered, infectious recombinant human p53 adenovirus particle, which restores the expression of this tumor suppressor.  It was approved by the Chinese food and drug regulators in 2003 for the treatment of head and neck cancer. Contusugene ladenovec (Advexin), a similar gene therapy, was developed by Introgen Therapeutics Inc and Contusugene Ladenovec Gendux  as an orphan medicinal product for the treatment of Li-Fraumeni cancer. However, Advexin failed to improve survival compared to conventional treatment and was turned down by FDA in 2008.

Onasemnogene abeparvovec was developed by the US biotechnology startup AveXis (which was later bought by Novartis) to treat spinal muscular atrophy (SMA) (a rare neuromuscular disorder manifested as the loss of motor neurons and progressive muscle wasting) and sold under the brand name Zolgensma. SMA  is caused by a mutation in the SMN1 gene encoding SMN, a protein necessary for the survival of motor neurons. The adeno-associated virus serotype 9 (AAV9) delivers the SMN1 transgene to the affected motor neurons, where it leads to an increase in SMN protein levels. Zolgensma is approved in the US and some other countries. 

Alipogene tiparvovec (the brand name is Glybera) is a gene therapy to treat lipoprotein lipase deficiency (LPLD), a rare recessive disorder attributed to a mutation in the lipoprotein lipase (LPL) gene, which can cause severe pancreatitis. The adeno-associated virus serotype 1 (AAV1) vector delivers a “healthy” copy of the LPL gene to muscle cells. It was approved by the European Commission in 2012.  Glybera was developed by researchers at the University of British Columbia. One of the leading researchers co-founded Amsterdam Molecular Therapeutics. Glybera became infamous as the "million-dollar drug" and proved a business failure. After spending millions of Euros on Glybera's approval, Amsterdam Molecular Therapeutics went bankrupt and its assets were bought by another company, uniQure

Alipogene tiparvovec treatment was expected to cost as much as around $1.6 million in 2012 (reduced to $1 million in 2015), making it the most expensive drug in the world at that time. However, replacement therapy cost approximately $300,000 per year. In 2017, uniQure announced that it would not renew the marketing authorization because of a lack of demand. As of 2018, only 31 people worldwide have received Glybera, and uniQure is not planning to sell the drug any longer.

Sitimagene ceradenovec is an adenoviral vector accommodating the herpes simplex thymidine kinase gene developed by Ark Therapeutics Group plc for the treatment of glioma patients. The European Committee for Medicinal Products for Human Use reviewed sitimagene ceradenovec efficacy data and considered them insufficient evidence of clinical benefit, so the marketing authorization application was turned down. 

 

Success 

Failure/cause

Gendicine

Gelsinger case/adverse effects 

Zolgensma     

Advexin/lack of effectiveness 

 

Glybera/price

 

Sitimagene ceradenovec/lack of effectiveness 

 

Although gene therapy is a promising treatment option for a number of diseases (including orphan diseases, inherited disorders, and some types of cancer), the approach remains risky as its safety and efficacy are not guaranteed. In addition, prices for such therapies are still too high to make them routine. Gene therapy is currently being tested only for diseases that have no other therapeutic options. 

We have considered only 1 mainstream of gene therapy - introducing a new gene delivered by adenovirus or an AAV vector to compensate a defective one. The development of adenovirus and AAV vectors has come a long way since their first use. As things stand now, there have been more failures and setbacks in this field. Does it mean that researchers have been discouraged from seeking new gene therapy options and ways to overcome the limitations? Definitely, no. For example, a 16-year company 

Sirion Biotech, which now is the leading European developer and manufacturer of viral vectors for research and preclinical applications, possesses one of the world’s most comprehensive viral vector technology platforms addressing all three major vector types – lentivirus, adenovirus, and AAV. 

Sirion Biotech`s patented bacterial artificial chromosomes (BAC) technology enables adenovirus construction in less than 5 weeks. “Tomorrow’s pharmacological achievements … will be driven by specialized genetic medicines that haven’t even been developed – yet!”  – says Dr Christian Thirion, CTO and founder of Sirion Biotech.

Topics: Novel Therapeutics   

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