1. Guest, the 'News in Brief' for the week beginning 18th January 2021 is here.
    Dismiss Notice
  2. Welcome! To read the Core Purpose and Values of our forum, click here.
    Dismiss Notice

Gene Therapy to Eradicate Herpesviruses

Discussion in 'Antivirals, Antibiotics and Immune Modulators' started by sebaaa, Jun 2, 2020.

  1. sebaaa

    sebaaa Established Member (Voting Rights)

    Messages:
    26
    Likes Received:
    190
    This is something that I wrote so that I can have a good overview on CRISPR/Cas9 and to also layout my thoughts in a logical manner since it’s often difficult to think with my severe brain fog. I hope that someone can find use for this and maybe suggest improvements if necessary.

    I think there is massive potential in CRISPR/Cas9 to potentially treat ME/CFS, or at the very least test hypotheses, even though the limiting factor will be an understanding of the underlying mechanisms of ME/CFS. If oral CRISPR/Cas9 is successful then I think that can have massive implications for medicine overall. Anyways...

    Viral infections have been an area of interest in ME/CFS for a long time, and have been theorized to produce the symptoms. The fact that Valcyte and antivirals work in some people reinforces the idea that viral infections could play a role in ME/CFS. One review article noted that while controversial there may be “potential viral involvement for at least a subgroup of ME/CFS patients [1].”

    The problem with this hypothesis is that it’s difficult to test by treating the problem. It’s clear that the viral infections can’t be lytic (active) otherwise antivirals would work quickly. Therefore, that leaves either latent infections (inactive) or abortive infections. But current antivirals only work for lytic infections.

    Gene Therapy

    This leads us to gene therapy, which could theoretically cure the human body of viruses by altering necessary genes, like ones necessary for HHV6 latency. Gene therapy has already been used for over a decade indicated by the 1000s of clinical trials and approved drugs.

    The most recent one, CRISPR/Cas9, will be the focus since it’s easy to use, cheap, and powerful. This is a rapidly expanding field and it already has heaps of in vitro and in vivo (murine) data, including data on herpesviruses. The first phase I clinical trial with CRISPR/Cas9 on humans happened in 2020, where they modified T cells in three patients with cancer [2]. The changes persisted for 9 months even though it didn’t cure their cancer.

    My goal will be to apply CRISPR/Cas9 technology to design an oral drug that can eradicate the body of EBV. However, this will require a bit of background knowledge...

    CRISPR/Cas9 Terminology

    CRISPR/Cas9, clustered regularly-interspaced short palindromic repeats, is a tool for editing the human genome [3]. There are 4 main parts to CRISPR/Cas9:
    • Cas9 protein: RNA-guided nuclease which produces double-stranded breaks at target sites
    • sgRNA: A single guide RNA that targets a DNA sequence
    • Cargo: The part that delivers the sgRNA and Cas9 protein into the cell
      1. DNA Plasmid
      2. mRNA
      3. RNP (ribonucleoprotein)
    • Delivery vehicle (vector): How CRISPR/Cas9 is delivered into the body. Either a viral or non-viral vector.
    Viral vectors, which are the most common ones, include the adeno-associated virus (AAV), adenovirus, and lentivirus [4]. Non-viral vectors include lipid nanoparticles, gold-based nanoparticles, and lipoplexes. Each one has distinct advantages and disadvantages [5]:
    • Viral vectors
      1. High transfectant (gene editing success) rate
      2. High immunogenicity
      3. High off-target gene editing
      4. Expensive
      5. Limited packing size
    • Non-viral vectors
      1. Low transfectant rate
      2. Low immunogenicity
      3. Low off-target gene editing
      4. Cheap
      5. Control over packing size
    Designing an Oral, Non-Viral CRISPR/Cas9 Drug

    Here we’ll be picking the most recent in vivo studies on non-viral CRISPR/Cas9 vectors. The focus on non-viral vectors is due to their low cost, which is helpful for experimentation, and safety. We will look at studies with other forms of gene therapy like RNAi to find out what techniques can make CRISPR/Cas9 available for oral administration. Finally, we’ll pick out the relevant sgRNAs from in vitro studies that used CRISPR/Cas9 to eradicate EBV.

    CRISPR/Cas9 Non-viral vectors
    • BAMEA-O16B (mice) [6]
    • TT3 (mice) [7]
    Oral delivery methods from other types of gene therapy
    • mannose-modified trimethyl chitosan-cysteine NPs (RNAi, rats) [8]
    • siRNA/gold NPs encapsulated in CS-taurocholic acid (RNAi, mice) [9]
    • Taurocholic acid coating [10]
    EBV targets for CRISPR/Cas9
    • BART promoter (latent) [11]
    • EBNA1, OriP, and OriW (latent & lytic) [12]
    Theoretically, to create an effective CRISPR/Cas9 drug for EBV that can be orally administered we can pick the mRNA (cargo) used in the BAMEA-O16B nanoparticle. We could use the ionic gelation and siRNA entrapment method to create the nanoparticle (vector), and replace the siRNA with the mRNA [8]. Finally, we use two sgRNAs to target the BART promoter gene.

    The ionic gelation and siRNA entrapment method is as follows:
    Alternatively, we can pick the BAMEA-O16B nanoparticle, and coat it in a glycol chitosan-TCA (taurocholic acid) solution [9]. Finally, we pick the appropriate sgRNAs to target EBV.

    Other Considerations
    • Manufacturing: There are many companies that do custom synthesis of sgRNAs like IDTDNA, Horizon, and Biolegio. The vector can be handled by the labs that synthesisze nanoparticles like nanoComposix, biosyn, and CD Bioparticles. The price is probably going to be around 1k but hopefully it is less.
    • Systemic administration: CRISPR/Cas9 can be administered locally to certain tissues but I think that making sure that it reaches most places in the body will be better so that it clear any viral reservoirs since I don’t have any information on where EBV resides latently other than in B cells.
    • Testing gene editing success: Knowing whether or not the gene editing actually happened in vivo is of some importance. In the in vivo trial on editing T cells, they developed custom assays to monitor safety. It seems this won’t be possible for some time at least.
     
    Studi and Peter Trewhitt like this.

Share This Page