Monday, March 05, 2012

Normal intracellular viral immunity- A problem in gene therapy?

Have you ever thought of this question…? “When a virus enters the cell the cell has to identify that there’s an intruder. Given the fact that the cell itself has a genetic material in it, the cell has to differentiate between foreign and self genetic material. So how does the cell do it? What is the implication when using viral vectors to deliver genes?”

The safest bet would be with DNA virus and the dsRNA virus. A normal cell would not have a DNA fragment (Am talking with reference to eukaryotic cell such as that in we humans), or rarely a dsRNA fragment. The dsRNA in normal cases will be taken care by host cells via pathways like RNAi pathway. (I will discuss about it in some other post. For a few basics, click here). The single stranded RNA would be more difficult to assess as we have similar types in our cells. However they have sufficient differences to be recognized.
Fig 1: TLR and RIG-I two antiviral innate immunity pathways.

So when a virus enters the cell the signals are detected by TRIF (TIR-domain-containing adapter-inducing interferon-β), MyD88 (Myeloid differentiation primary response protein 88) and RIG-I (Retinoic acid inducible gene I, also known as DEAD-box protein 58). For an excellent review article on the mechanism please refer to the review by Rashu B Seth et al. Newer signals and responders are identified, an active field of research. 

No matter how the pathway starts, the signaling cascade culminates in MAVS. The mitochondrial anti-viral signaling protein (MAVS), or otherwise known as CARDIF, IPS-1, KIAA1271 and VISA, is mitochondria associated protein that regulates type I interferon through a well structured and highly regulated production of NF-kappaB and IRF3. The N-terminal CARD domain of MAVS interacts with RIGI.


Fig 2: DNA Sensors and Anti‑Viral Immune Responses.

The CARD domain stands for Caspase recruitment domain. Caspases are enzymes that are involved in Apoptosis regulation in cells.

Genetic diseases are a group of inborn errors that can clinically vary depending on the gene affected. Loss of a gene function can be corrected by injecting the cell with a functional DNA and incorporating it into the genome (Gene therapy). Who else can deliver a gene to the cell better than a virus in natural conditions? There are many ways to achieve a gene therapy procedure. Major protocols under study include 
  • Cationic liposomes 
  • DNA condensation and receptor mediated gene transfer 
  • Viral vectors such as Retrovirus, Adenovirus, Adeno-associated virus etc 

Fig 3: AAV life cycle, Shyam daya etal
Adeno-associated viruses are small single-stranded DNA viruses of the Dependovirus genus belonging to Parvoviridae. AAV-2 is used in experimental studies. AAV-2 enters the cell through an endosomal route, is transported to the nucleus by motor proteins, and enters the nucleus, everything happening in a flash.          
So now that you have understood the background from various angles, let me put things together. This blog post is based on the paper, “Innate responses to AAV vectors” by Rogers et al. This paper looks at the hindering effect of innate immune response to the AAV vectors which has the potential to deliver important genes.

The paper starts with an introduction to the AAV importance as a vector in gene transfer and also some current perspectives of the innate immunity to virus in a cell. The sensors detect the arrival of an extracellular gene leading to multiple phosphorylation event (This is common. Most of the intracellular signaling is based on phosphorylation or de-phosphorylation of various intermediates). The pathway culminates in stimulation of NF-κB Classical pathways.

Fig 4: A proposed model for innate immune
recognition of AAV vectors
The second part of the paper focusses on the Specific response to a AAV. Here it was noted that the ssAAV (Single stranded AAV) typically produced less immune response compared to other Adenoviral vectors, and the scAAV (self-complementary AAV) produced more response in comparison to the ssAAV.

A model was also proposed to show the possible mechanism of innate immune recognition of AAV vectors.        

The last part of the paper is dedicated to its implication in clinical practice.

The AAV is a very good vector of choice for treating genetic diseases as it provides a flexibility at various levels. The problem is that the viral matter can elicit a immune response, which often is low, but good enough to have some tissue degradation. That may lead to treatment failure. So the possibility of use should be supported by some antagonists of these response.
Further Reading:

Rogers etal. Innate responses to AAV vectors. Frontiers in Microbiology. September 2011; Volume 2 Article 194

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