A new advance in gene therapy

Introduction

When I say viruses what is the first thing that comes to your mind? Its most probable you went ick! You might have even imagined a pandemic or an unwanted trip to the doctor’s office. While this is certainly in the realm of capabilities for viruses, it does not demonstrate the full breadth of their potential effects on us either. In fact, contrary to popular knowledge certain applications of viruses can be beneficial to our health if used correctly. Imagine cancer for instance, a disease which is caused by mutations in the DNA leading to rapid cell growth resulting in greater physiological issues. Sometimes cancer can be very hard to treat and unfortunately leads the numerous deaths globally each year [2]. While new drugs and chemotherapy treatments are helping to reduce the number of fatalities, they still fail to address the genetic root of the problem. This is the major dilemma of current cancer treatments but, what if we could procure a treatment method which allows us to target cells on the genetic level instead of just the physiological and cellular. This is where the potential of viruses to benefit our health truly shines with the introduction of the concept of gene therapy. Gene therapy is the use of genes as a therapeutic agent to replace the bad or deleterious genes within cells which lead to development of diseases. The requirement of a delivery system known as a vector is essential in the practice of gene therapy in order to deliver the therapeutic gene to the targeted site. Viruses are well suited for this task as they are designed by nature to complete this exact task as a means of their propagation and survival. Billions of years of evolution have granted viruses the ability to evade the immune system, transfect (inject or release its genetic payload), and insert itself into the host DNA code. Several viruses exist and are currently undergoing study for use in gene therapy treatment. Of them however, Adeno Associated Virus (AAV) is among the most well studied for use at this task. One major problem exists however, while viruses are excellent at evading the host immune system AAV is not particularly well suited in this regard about 70% of the human population gains an anti-AAV immune response very early in life that limits AAV’s potential as a viral vector [5]. In contrast however AAV’s non-pathogenic nature and its ability to predictably join into specific parts of the host DNA in a way which can be tracked and monitored, as well as its numerous variations which are specified for an array of cell and tissue types still makes it a competitive choice compared to many others. In this blog post we will discuss a method attempted by our lab in order to create and AAV vector which can evade the host immune system while maintaining its attractive benefits.

Method

   AAV is a small virus and correspondingly has a small DNA code. In terms of speaking about the simplicity of the virus’s DNA code it is only comprised of two separate genes known as REP and CAP. These genes encode for molecular components known as protein which help to ensure the virus has everything it needs in order to reproduce itself successfully. The REP gene encodes these proteins exclusively for genes that control replication of AAV’s genome code while the CAP gene codes the proteins which make up the virus’s protein coat. The research we have conducted in the past semester on AAV has solely focused on the CAP gene as the protein coat which it encodes for makes up the part of the virus which is specifically affected by the anti-AAV immune response. Furthermore, when we are discussing the immune response mounted against AAV via targeting of its protein coat we are referring to its recognition by antibodies, protein molecules which serve as signals so that cells involved in the immune system can see and get rid of the virus that is present. Since the destruction of the AAV virus depends on the recognition of the virus by antibodies we decided to develop a way for the AAV protein coat to essentially hide itself from recognition. In order to potentially achieve this, we ever so slightly altered the DNA sequence within the CAP gene. We performed this alteration in a way which allowed us to randomly alter specific areas of the CAP gene, making numerous variations of it in a short period of time. We targeted these areas based of structural data previously provided in scientific literature from previous studies outside of our lab [. Removal of the CAP gene DNA sequence from that of AAV DNA sequence was required as the first step for us to see if we could attain the desired result. Additionally, we fragmented the CAP gene into separate pieces which made the areas that we targeted for alteration more confined than in previous attempts making attainment of the desired alterations more likely. Following the removal and alteration of the CAP gene sequence we created what is known as a DNA library of these new variations of the CAP gene, placing it back into the AAV DNA code and allowing the virus to grow in cell culture. Our new novel AAV variants were then tested against antibody A20 in order to see if it could evade its binding. We selected A20 for the purpose of our study as previous research indicated that it bound the same region of the AAV protein coat many other antibodies involved in the anti-AAV immune response bound as well. As such the prevention of its binding to the AAV protein coat would in theory create an AAV virus that could evade several other anti-AAV antibodies as well [3]. A column containing millions of small resin beads with A20 bound to it in a stationary fashion was created to test their ability to evade A20 binding. Our novel variants purified, were applied to the column and our selection was made on the previously stated basis. The novel AAV that did not bind to the column ended up in a sample known as the flow through where they were all captured. Though able to attain AAV which was able to evade A20 was achieved, this only answered one portion of the dilemma in the use of AAV for gene therapy. Concerns about alteration to an area of the AAV protein coat known as the deadzone were also had. Preservation of the deadzone is extremely important for AAV to be able to transfer its therapeutic gene to the targeted area when used as a viral vector. In previous studies using a less targeted method alteration to this region lead to poor gene delivery to the targeted area when this area of AAV was altered. To insure deadzone integrity in the novel AAV variants which were able to evade A20 binding we tested their ability to bind their primary receptor required for viral entry. The ability of our mutants to still be able to bind these receptors is significant as it was found that in studies previously mentioned this binding was not achieved. Before we were able to do this however a method allowing us to separate each novel variant individual was applied in order to test their binding individually. We utilized the virus’s latent life cycle and a pseudo plaque assay as a means of identification in this regard using fluorescence to identify each individual [1]. This method reflects the ability of the individual AAV variants to interact with its specified cell types. As the method relies on fluorescence, those individual variants which gave such off demonstrated some level of efficient entry. However, we wanted to obtain real quantitative data to determine which individual variant was most effective in this regard. To do this we applied a final separate method using solely the receptor and antibodies not related to the anti-AAV immune response to quantify this ability. Known as an ELISA this assay utilized a plate with the receptor fixed to the bottom of the plate. Introduction of our AAV selected from the pseudo plaque assay were applied to the plate and allowed time to fix to the primary entry receptor. We then apply the non-related antibody to the plate and allow it to attach to the AAV protein coat. We can then quantify this based on the release of a fluorescent tag which is released from the antibody one it binds to our AAV variants. The amount of fluorescence calculated from each part of the plate where each individual AAV variant is separately gives us an idea of which variants likely have the best entry and thus potentially the best delivery of therapeutic agents to targeted areas. The higher the absorbance the better the AAV variant is in this ability. Lastly, we made our selection of the best AAV variant we created based on these criteria and once more grew it up for later sequencing and use in clinical trials.

Outcomes

    While this is not necessarily yet conclusive it does give promise for the potential of a new AAV variant which can be used in clinical trials. To add to this what is extremely important about what we have managed to create in our study is that is based upon the most common natural variant of AAV which is known as AAV-2. This is the most well studied and widely used natural AAV variant and as such if future data and studies prove optimal could find its way to wide-spread use [4]. What’s even more important for me in you if use of our vector becomes a reality is that we will attain a way to treat diseases like never before. Imagine a future where we can treat diseases once thought to be inoperable and incurable, this work could make this possible. This could increase human longevity and even further propel our understanding of how these diseases work. Additionally, more efficient ways to conduct gene transfer would be very useful in biotechnology in general increasing the speed at which new information is attained and applied for the benefit of us all. Therefore, we hope to continue with this work and achieve all of these goals for a brighter tomorrow!

References:

1. “Application of the Pseudo-Plaque Assay for Detection and Titration of Crimean-Congo Hemorrhagic Fever Virus.” Journal of Virological Methods, Elsevier, 10 Aug. 2012, www.sciencedirect.com/science/article/pii/S0166093412002777.
2. CDC – Expected New Cancer Cases and Deaths in 2020. (2015). Retrieved July 28, 2019, from https://www.cdc.gov/cancer/dcpc/research/articles/cancer_2020.htm
3. Chapman, M.s., and D.m. Mccraw. “Structure of Adeno-Associated Virus-2 in Complex with Neutralizing Monoclonal Antibody A20.” 2012, doi:10.2210/pdb3j1s/pdb.
4. Lochrie, M. A., et al. “Mutations on the External Surfaces of Adeno-Associated Virus Type 2 Capsids That Affect Transduction and Neutralization.” Journal of Virology, vol. 80, no. 2, 2005, pp. 821–834., doi:10.1128/jvi.80.2.821-834.2006
5. Mingozzi, F., and K. A. High. “Immune Responses to AAV Vectors: Overcoming Barriers to Successful Gene Therapy.” Blood, vol. 122, no. 1, 2013, pp. 23–36., doi:10.1182/blood-2013-01-306647.