Viral gene therapy (scientific)

By: Eunice Lozada-Delgado

Gene therapy is a promising technique that is being studied to treat different diseases related to gene mutations. Some diseases such as muscular dystrophy or cancers are caused by gene mutations, thus the most effective way of treating them would be by correcting said mutations. Gene therapy focuses on the delivery of a specific gene(s) to targeted cells with a mutation or loss of this gene so that the gene product can be restored. Furthermore, since direct insertion of genes to a cell generally does not function, a carrier for this gene should be used. Carriers being studied today are viral vectors. Viral vectors because viruses are known to infect target cells and insert their genomic information into the host cell successfully. Therefore, viral vectors are currently being genetically engineered to carry the desired gene without causing disease.

There are different viruses being used in studies as viral vectors. Most are of retroviruses and adenoviruses but also adeno-associated viruses, herpes viruses, lentiviruses, among others also being studied for gene delivery. The difference between all of them lies in: whether they alter the target cells genetic material temporarily or permanently, how well they transfer the genes, and how they infect their target cell (genetherapynet.com). Some examples of related recent studies and their possible applications are going to be briefly discussed next.   

First, in a study by Lostal et al. they were trying to use an Adeno-associated viral vector (AAV-vector) to carry the dystrophin gene into Duchene muscular dystrophy (DMD) mice (Lostal et al., 2014). The problem they found was that this carrier viral vector has small packaging capacity of carrying  up to 5kb while the dystrophin cDNA is >11kb. Therefore, they engineered various sets of what they call tri-AAV vectors, where they split the dystrophin cDNA into three pieces independently packed into three recombinant AAV- vectors.  Then they tested their efficacy of insertion and expression of the full dystrophin gene after injecting DMD mice with all three vectors together. They found that even with low reconstitution efficiency, expression of the full dystrophin gene was found in the muscle tissue. In essence, they were able to successfully express a split recombinant gene carried by AAV-vectors into DMD mice. This also brings light into the possible use of the delivery of large genes using tri-AAV-vectors. Now, further studies have to be made to optimize these findings as well as evaluate disease progression.

A similar study in DMD follows in a video: 
https://www.youtube.com/watch?v=S7gFK6w_3Q0

Furthermore, another study by Tardieu et al. focuses on a Phase I/II trial of Mucopolysaccharidiosis type IIIA  patients using Adeno-associated viral vector (AAV-vector) (Tardieu et al., 2014). Mucopolysaccharidiosis type IIIA is a degenerative disease caused by a mutation on the gene encoding the N-sulfoglycosamine sulfohydrolase (SGSH) which is activated by a sulfatase-modifying factor (SUMF1). In this study they chose 3 patients of 5.5-6 years old and one of 2 years and 8 months old. These patients received intracranial injection of AAV-vector encoding both human SGHS and SUMF1 cDNAs together with immunosuppressive treatment for better response, and where followed up for 1 year. At this point they already have data of the use of this vector in mice and dogs, thus now are going to this phase I/II trial in humans. After injection, they were evaluating if these patients had any immunological response, secondary effects as well as cognitive benefits due to the treatment. What they found was that the treatment was relatively safe for that year, the largest symptoms in the patients where diarrhea that was able to be controlled. In terms of the cognitive benefits they were most observed in the youngest patient that didn’t have brain atrophy as the other three older patients before treatment. They conclude that in this first clinical trial they observed some improvement together with safety which could lead to further clinical trials with increased vector dosage and additional injection sites to test.

In summary, in this entry we have discussed various recent studies in human viral gene therapy being used for the treatment of diseases as Duchene muscular dystrophy and Mucopolysaccharidiosis type IIIA. As demonstrated, these studies are currently being done not only at the animal model level but also some have made it to human clinical trials. Even though great advances have been made recently, more research is needed to optimize these promising efforts to be able to efficiently and safely treat mutation based diseases with these viral gene vectors.

References used:

LOSTAL, W.  et al. Full-length dystrophin reconstitution with adeno-associated viral vectors. Hum Gene Ther, v. 25, n. 6, p. 552-62, Jun 2014. ISSN 1557-7422. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/24580018 >.

TARDIEU, M.  et al. Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with mucopolysaccharidosis type IIIA disease: results of a phase I/II trial. Hum Gene Ther, v. 25, n. 6, p. 506-16, Jun 2014. ISSN 1557-7422. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/24524415 >.

Viral vectors information, recovered 3/29/15 <http://www.genetherapynet.com/viral-vectors.html>

Exome sequencing analysis of Retinitis pigmentosa

By Edgardo Lopez

Retinitis pigmentosa (RP) is a degenerative disease that is characterized by blindness and retina spots formed by pigments that blocks the cornea. This disease doesn’t show during early stages of development until you pass puberty. In a genetic aspect this disease is consider a rare disease since there are many genetic possibilities to obtain it from your parents as an autosomal dominance (adRP), autosomal recessive (arRP), X-linked (xlRP), or even transmitted as a mitochondrial or digenic trait in the most rare cases.  The RetNet database has report that there are 62 genes that are associated to RP. Some of those genes are mutations or defected variants of other genes like RHO, RPGR or USH2A to gave some examples. For a better reference of how this disease behaves, a group of scientist analyzes the frequency of mutations and genes that are responsible of RP in a population in China. Performing better analysis with emerging technique like next generation sequencing will provide a faster way to achieve a better molecular diagnostic of the disease in the patient or even on a population (like groups of families). In this study they use Whole Exome sequencing (WES) that basically is the nucleotide date obtain from the whole genome protein coding section to detect variants in 62 causative genes from 157 unrelated Chinese families with the disease. To perform the WES this group of scientist use blood samples from leukocytes of peripheral venous to obtain the genomic DNA. NimbleGen SeqCap EZ Exome system was use to obtain the Exome library from the DNA samples and Sanger sequencing also was used to confirm the gene variants that were identified by WES.

An example of a retina without the disease and with RP(picture A) and also how a person with RP sees through time(picture B). 

A

B

As a result from the WES analysis 244 candidates variants were detected from 60 of the 62 genes that are highly related to RP and 240 of those candidates were confirm also by Sanger sequencing. From the bioinformatics analysis 50% of the variants that were identified are novel to be consider potential pathogenic mutations. More that 30% of the mutations were link to heterozigocity associated with adRP, more than 40% of homozygous or compound heterozygous mutations associated with arRP, and less that 15% were hemizygous mutations associated with xlRP. The most frequent genes found in the 157 families were RHO, USH2A and RPGR that are found in adRP, arRP and xlRP.

In the following picture you can see the proportions of individual genes on the 157 families.  

For some strange cases of RP that doesn’t have that same behavior, future studies are expected to include possible pathogenic variants in noncoding regions and deletions in the whole Exome.  Using next generation sequencing helps to achieve great results in this article to determinate pathogenic mutations in RP patients that other techniques wouldn’t and approximately 50% of the population appears with the 60 genes that has been detected in previous studies of RP. This article shows a better understanding of the relationship between genotype and phenotype in a certain disease that can be implemented in many other for future medical goals.

Reference:
Yan Xu et. al. 2014 Mutation of 60 known causative genes in 157 families with retinitis pigmentosa based on exome sequencing. Journal of Human Genetics doi:10.1007/s00439-014-1460-2

  

Clinseq: Genome profiling for a better personalize health treatment. (Non-scientific)

By Edgardo Lopez

Imagine a clinical program that will help you to prevent, diagnose and treat a disease like cancer o a cardiovascular disease by using your DNA information? A group of scientist led by Dr. Leslie G. Biesecker develop a project were the genome takes a big role in the discoveries of treatments for different diseases. One of Clinseq project main goal is to use whole genome sequencing to have a better DNA profile of each patient compare to there family and other patient with heart disease, breast cancer, and hearing loss. This study also want to explain how genetic variation affect our health, present the genetic results to the people and also create better ways to store genetic data and analyze the complexity no matter what. In this study they work with a cohort of people with a background in Atherosclerosis heart disease.

¿What is Atherosclerosis heart disease?

This a disease in witch your plaque block your arteries causing major problems like heart attacks, strokes or even death.

By elucidation the human genome we have discover new advances in DNA sequencing technologies and new ways to interpret the genetic architecture of the DNA related to some diseases. Using the whole genome is a better approach to understand the variability and complexity of the genome and by taking multiple gene analysis we can get a better point of view of what is expressing in our cells and what not. The design of this study is base on an analysis of 1000 individuals in the National Institutes of Health. Taking DNA samples for sequencing is one of the most important steps in this project since there are issues in the effectivity of the data obtained by sequencing. Each individual will get a multiple analysis of variant that will collaborate with the clinical sector for a genetic and medical counseling.  Clinseq is one of the most important approach for medical science that use whole-genome analyses and a clinical approach. For each patient there is a enrollment process to participate of this program. Each patient need to provide a clinical background of the atherosclerotic hears disease in their family and also in them. DNA samples are taken for clinical evaluation and for research. The research samples pass throw a bioinformatics analysis and then is validate with the clinical data to learn and discover the relevant information that can lead to a better treatment for the patient.

In this video you can take a more personal view of the project and their patients.

Reference:

Biesecker et. al. 2009. The ClinSeq Project: Piloting large-scale genome sequencing for research in genomic medicine. 19:1665–1674; ISSN 1088-9051/09; www.genome.org

What is Atherosclerosis? NIH National Heart, Lungs and Blood Institute http://www.nhlbi.nih.gov/health/health-topics/topics/atherosclerosis