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Gene Editing: A Review of CRISPR/Cas and the Challenges to Overcome

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A many of you know, CRISPR/Cas is a gene editing technology that many hope to have cure a plethora of viruses and other genetic issues, including HSV.  Drs. Bryan Cullen and Edward Kennedy have put together a paper that discusses that status of the CRISPR/Cas technology.  Their paper is unpublished but will likely appear in January 2017.  I've taken a few snips from the paper that would be of interest to us here.  While there is some good information, I think the authors fall short on identifying all the issues with the technology in relation to HSV application.  Still, it reinforces what many of us already know.  Enjoy.

https://www.ncbi.nlm.nih.gov/pubmed/27576009

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Although the use of some form of liposome or nanoparticle is potentially feasible, the best-studied method of in vivo gene delivery is viral vectors, especially lentiviral vectors and vectors based on adeno-associated virus (AAV). In the context of Cas9 delivery, lentiviral vectors have the major advantage that their packaging size of up to ∼7 kilobases (kb) readily accommodates the SpCas9 gene (∼4.2 kb) as well as one or more sgRNAs and the cis-acting regulatory sequences required for efficient expression. However, titers of lentiviral vectors cannot exceed ∼10 (to the 9) infectious units per milliliter, which is not adequate for the efficient infection of an entire population of virally infected cells in vivo (10), such as HIV-1-infected T cells, even though lentiviruses are highly lymphotropic. In contrast, AAV-based vectors can be generated at titers up to 10 (to the 14) genome equivalents per milliliter. However, two limitations stand in their way.

Continued..

Quote

AAV has a packaging capacity of only ∼4.8 kb. This makes it impossible to express the ∼4.2-kb SpCas9 gene, together with an sgRNA, from a single AAV vector. One approach is to use two AAV vectors: one to express SpCas9 from a minimal pol II promoter element while the other encodes one or more sgRNAs (11). A second approach is to use a different, smaller Cas9, for example, the ∼3.2-kb Cas9 gene encoded by Staphylococcus aureus (SaCas9) (12, 13). SaCas9 has a somewhat less common PAM (5-NNGRRT-3, where R is purine) but otherwise appears to have a cleavage capacity and targeting accuracy comparable to SpCas9. Single AAV vectors able to express SaCas9 and one or two sgRNAs have been described and appear potentially very useful for in vivo gene editing (12, 13). A second concern with AAV vectors is their limited tissue tropism, although this has gradually expanded with the identification of additional AAV variants from different species and the derivation of AAV recombinants with enhanced tropism for specific tissues (14). AAV serotypes with a strong tropism for hepatocytes, neurons, and epithelial and endothelial cells have been described, but AAV variants able to efficiently infect lymphoid cells remain to be identified, which is a particular problem if editing of the HIV-1 genome is envisioned (see below).

One final issue with viral vectors relates to their potential to integrate and, hence, cause mutations that could inhibit or enhance the expression of specific cellular genes. Lentiviral vectors include integration as a key step in their life cycle, so this is clearly a concern. In contrast, AAV vectors do not normally integrate into target cell genomes, but integration of wildtype AAV (2) has been reported in a small number of hepatocellular carcinomas (15). Whether this represents a real concern with AAV vectors, which have gained wide popularity as tools for introducing genesinto hepatocytes in vivo, remains to be established.

In considering which viruses might represent appropriate direct targets for elimination using CRISPR/Cas, two criteria are paramount. First, the virus must be a DNA virus or exist in a DNA form as part of its life cycle. Second, the virus must replicate in a discrete, well-defined region of the body that can be accessed by vectors expressing a virus-specific Cas9/sgRNA combination. In our view, the list of potential target viruses (Figure 2) is therefore rather limited and consists primarily of hepatitis B virus (HBV), human papillomavirus (HPV), herpes simplex virus (HSV) types 1 and 2, and human immunodeficiency virus type 1 (HIV-1).

HSV 1 and 2 specific

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In latently infected neurons, HSV-1 and HSV-2 genomes are found as circular, nonreplicating DNA episomes in the cell nucleus with from 1 to perhaps as many as 50 copies per latently infected cell (28). These dsDNA molecules are in principle excellent targets for cleavage and either destruction or mutational inactivation by CRISPR/Cas (29–31) (Figure 2). Moreover, because the likelihood of reactivation of HSV-1 or HSV-2 directly correlates with the viral DNA load in the ganglion, even an incomplete ablation of the pool of viral genomes might have significant benefits. Neuronal cells are good targets for infection by AAV, and it seems possible that AAV-mediated delivery of SpCas9 or SaCas9, together with possibly multiple sgRNAs specific for HSV-1 or HSV-2 to shatter or mutationally inactivate the viral genome (Figure 2), would represent a potential strategy to cure this bothersome disease. Indeed, recent data suggest that HSV-1, even during lytic infection, may be an excellent target for treatment using a CRISPR/Cas-based approach (32).

 

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Atrapasueños

El Dr. Halford y su vacuna esta teniendo poca credibilidad, y ahora bryan cullen empieza desde 0 después de que en 2008 dijo que estaba apunto de curar el herpes que está pasando todos los ensayos se están desmonorando  y los científicos están bajándose del barco 

 

Dr. Halford and his vaccine are having little credibility, and now bryan cullen starts from 0 after that in 2008 he said he was about to heal the herpes that is going on all the trials are being dismantled and the scientists are getting off the boat

 

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Atrapasueños

Study after study has shown that when clinical trials involve entities with a financial interest in the outcome, as the Parker Institute for Cancer Immunotherapy and Penn have in this one, the reported outcomes are more likely to be favorable than when the trial is sponsored by, say, the National Institutes of Health. In studies where the sponsor has a profit motive, scientists are also less likely to adhere to best practices, research has shown. “If you really believe in a [bio]technology and it’s not completely clear whether a side effect is the fault of the disease or the technology, your bias could influence how you interpret that,” said Atkins.

In 1999, members of the Recombinant DNA Advisory Committee pointed out, a young man died in a now-infamous gene therapy trial at Penn in which the lead scientist had a multimillion-dollar financial stake in the technology. That conflict of interest, scholars have argued, may have led him to make dangerous decisions. Although the Parker Institute will handle patents for any discoveries that emerge from the research it funds, “each site owns its intellectual property,” said chief legal counsel Melinda Griffith. “If you invent it, you own it.”

Or, everything could go well and CRISPR cures cancer.

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Evaluate

It's difficult to understand your translations often times. I'm not really sure what to say to many of your posts since the context is difficult to interpret.  I can imagine that is frustrating for you and I hope that somehow that is overcome.  What I will say is if you quote any study, paper or article you should provide a reference for that quote.

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