CRISPR : A tool for genome editing

By Edgardo Lopez

            Streptococcus pyogenes is the bacteria that were discover that has the capacity to knockout exogenous genes when is infected by a virus through non-homologous end joining (NHEJ). This adaptive mechanism is call CRISPR (Clustered Regularly Interspaced Palindromic Repeats) and in recent years this tool is been use for sequence-specific editing of mammalian genome. This knockout tool has been very efficient in NHEJ-induced indel mutations but in point mutations or sequence fragments directed by a homologous template is bean poorly efficient. In this article I’m discussing the new approaches to this tool and the optimization in the technique for a better genome editing. CRISPR is a great technique to achive genome editing for both biomedical research and clinical application s like possible specific knockout of genes that are related to a certain disease. Since this technique is practically new it have some poor performance in the specificity of editing. Chen Yu and his colaborators had found four small compounds that can enhance or inhibit CRISPR. The two enhancers are called L755507 and Brefeldin A. L755507 is a beta 3-adrenergic receptor agonist that increased the efficiency of gene insertion and Brefeldin A is an inhibitor of intracellular protein transport from the Endoplasmic Reticulum to the Golgi apparatus. These two enhancers are now known as molecules that significantly improve the insertion fo new genetic information into the cell.  The two inhibitors are called azidothymidine(AZT) and Trifluridine(TFT) and both of them are thymidine analogs. AZT is also use as an anti-HIV drug inhibiting reverse transcriptase and TFT as an anti-herpes virus drug that blocks DNA replication. These two inhibitor of insertion are now well known in this process, but they also enhance deletion of DNA theorizing that the two processes are competitive actions in the cell Through the process of validation this group of scientist perform cytotoxicity analysis, plasmid transfer to different mammalian cells, chemical screening to see activity, validation of the enhancing and repressing compounds using flow cytometry analysis and analysis of gene insertion.

The development of high-throughput efficient compounds form CRISPR genome editing has result in a better way to interact with NHEJ or homology-direct DNA repair pathways. The identification of the chemicals doesn’t exhibit high toxicity and works great in different types of cells. Upgrading genome-editing tools can lead to better ways of insertion, replacement of even removal of DNA in human cells. By mentioning those approaches I’m looking forward in the future  for new drugs that can be more personalize and more efficient and specific.

Reference:

Chen Yu, et. al.  2015.  Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells. Cell Stem Cell 16, 142-147 http://dx.doi.org/10.1016/j.stem.2015.01.003

WHOLE GENOME SEQUENCING MAY IMPROVE PATIENT MANAGEMENT (SCIENTIFIC)

 By CESAR PEREZ

Different diseases have different phenotypes, but there are some diseases with remarkable differences inside the same problem. Consequently, there is a difficulty in detection and treatment. Bainbridge and his team presents a study were they show advantages on the using of whole genome sequencing in the search of variants inside people's genome in order to detect high frequency variants for discriminating heterogeneous diseases from another kind of problems or variants that are benign.

For the study described, the scientists focused on Dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD), a disease with Mendelian inheritance. DRD is a clinically and genetically heterogeneous disorder which makes it ideal for searching the coverage of whole genome sequencing as a clinical tool. However, there is a difficulty on the diagnosis of DRD which is symptoms variation along the day, it is very common to find reduced dystonia by awakening and increasing distonia during midday. Traditionally, differential diagnosis for DRD was applied including  early parkinsonism and cerebral palsy. On the other hand, clinical diagnosis is focused in a neurological scope, inheritance, age, presence of certain metabolites, and the response to L-dopa treatment.

Scientist realized a whole genome sequence of twins diagnosed with DRD, its unaffected older brothers, unaffected parents, and cousins. Then, all the genetic data was analyzed in order to found single nucleotide polymorphisms (SNPs). In addition, affected twins were treated with L-dopa and were monitored. Among the results obtained, a set of putative nucleotides were found which were probably related to DRD. In order to validate the variant sites, a set of primers was developed, then PCR was realized. Actually, designed primers were able to detect the mutation in affected twins, and parents or relatives. Finally, Twins were treated, and after 4 months of treatment presented improvements in focusing in school and  athletic activities.

This work was important due to the power of whole genome sequencing could help in the diagnosis and treatment of DRD, plus another highly heterogeneous symptoms diseases. Sequencing methods not only could be used to diagnosis, but also to found risk on inheritance of different disorders. In addition,  the analysis of genomes presents more and more advantages like less time consumption, analysis of the whole picture of a disorder, or the discovery of new sites related to diseases. Nevertheless, this technique must face challenges yet: information among the patients,  unknowing variants and functions, undercovering of the relationship between variants alleles and phenotypes, or the develop of practical medical databases for the variants' information.

References:

Bainbridge, M. N., Wiszniewski, W., Murdock, D. R., Friedman, J., Gonzaga-Jauregui, C., Newsham, I., ... & Gibbs, R. A. (2011). Whole-genome sequencing for optimized patient management. Science translational medicine, 3(87), 87re3-87re3.

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>