I am sure no one these days is unfamiliar with the words CRISPR and gene editing. Come on, it's all over the news. They removed HIV genome from the mouse, they fixed a genetic mutation in the dog. Even more interesting are we close to designing human babies. Right? But have you ever though that what will carry CRISPR and Cas9 genes to the cells in the body that needs fixing?

Choosing the right transport for delivery of CRISPR-Cas
Illustrated by @scienceblocks using -
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Previously on CRISPR

In case, if you have not heard about it do read my previous blog. In that blog, I describe what CRISPR is - an adaptive immune system of bacteria against viruses that infect them. I also talk about how we used the bacterial immune system and designed it to be used inside mammalian cells for our selfish motives. Finally, you will find examples of how we have been exploring the potential of this new technology and what all could we possibly do with it. Though, for current discussion it is not really important that you understand functioning or CRISPR technology, but if you want to understand it in detail do read that blog.

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Yes, using CRISPR we can fix mutations, cure incurable diseases like HIV and even make a human shine green if we want. Nevertheless, there is this huge rate limiting step holding us back. And it's not just about CRISPR technology. The problem has also slowed the progress of gene therapy overall, the buzz word of last decade. Delivering CRISPR-Cas9 gene is just a part of gene therapy, where instead of gene you deliver your DNA sequence which has genes for gRNA and Cas9. The problem is of finding a safe and efficient transporter for delivering genes to the target cells, inside the body of a live animal(major concern: Humans). In this post, I will take you through some methods that exist to deliver the genes. We will talk about their pros and cons. Where are we headed? Then, in comment section we can also brainstorm ideas to find novel solutions(let's see).

Honey, I modified the kids!

Lights, Camera, Lipofection...

The positively charged liposome binds to DNA and transports it inside the cell via endocytosis
Doodled by @scienceblocks

A method I use on everyday basis to insert the genes inside the cells is pretty simple. What happens when you put oil in water and shake that damn thing? It forms droplets, right? Well that's because lipids don't like water, they like to be in company of the like molecules other lipids. Now say I take amphiphilic lipids - which has one charged group such as a phosphate or amine group(this end polar and loves water), attached to a long chain fatty acid(the chain part hates water). Yeah, same phospholipds like things that make cell membranes. However, for our purpose we will use the positive charge lipds such as those with amine groups. Then, I put a hell of such molecules in water. Want to guess what happens? They form tiny spherical particles called liposomes, which in their core have the chain part, while they expose their charge group to the surface.

Now comes the fun part. The DNA has negative charge on its surface(the phosphate backbone). So what happens if I put DNA and our positively charged liposomes together? You got it. They stick together! Now, a you need to do is put these liposomes-DNA complexes you made on the cells, and cell would pull it inside via their natural process called endocytosis₁ . Once inside the cell, your gene of interest will be expressed and do what it was supposed to do(or at least you will hope so!).

Now cells are fine, and it won't even hurt you use liposomes to transfect fertilized zygotes of the animal; however, what about adult live animals? Isn't that where we would be most interested in to treat diseases. Well the first challenge will be out of trillions of cells in the human body you would want to target specific cells. Now that is not hard to achieve. In fact you can just attach the antibodies specific for cell type or even some other specific molecule to the liposome. However, the major issue over here is the degradation of liposomes inside the body. Well it's pretty high. However, to overcome this Son et al., 2000₂ modified the surface of liposomes with other polymers, such as Polyethylene glycol(PEG). Though, PEG is not the only polymer to be used. In fact, since then many other formulation of liposomes have been tried, not only in lab but also in clinical trial₃
.

There are few safety issues. However, most clinical trials with liposomes have been disappointing in terms of their efficiency. And, this is an issue that even I face even when trying to transfect primary cells(cells derived from animals as opposed to cell lines), in vitro. However, we have not given up this idea. The research is still being carried out to get maximum safety and efficiency from these particles. Some researchers such as Kurrikoff₄ have developed a similar system using cell penetrating peptides. What I think is cool part about it? Well first peptides can be designed and modified with much more control and highthroghput. However, how efficient they are? Well, only clinical trials will tell us more.

Let's be direct?

But talking of efficiency microinjection₅
comes to mind. As the name suggests method is straight forward. You put a very fine needle like thing inside the cell and insert the damn gene into it. I mean what could be more direct. Well this would have been perfect, if it was a single cell that you are intetested in or even for making designer babies. But, if you have to deliver a gene to a live animal, say a CRISPR-Cas system to remove HIV genome, how many cells are you going to inject. So a good idea, but could be very impractical for what we desire. So is there a way to specifically and efficiently target many cells? Yes there is!

Meet viruses

I mean when it comes to being specific and yet robust, who are we to question a virus(not that it would answer anyway). Viruses not only are specific to hosts, but also to cell types they infect in the host. Moreover because we can mutate, modify and select virus surface proteins and per our whims we can play with both very specific and broad range of cells. The type of viruses in candidate list for gene therapy ranges from using adenoviruses, herpes viruses to lentiviruses. Of this my favorite are adeno and lenti because they are most commonly used by not just me but many others in the labs around the world. So, I will talk about them, however if you want to read more about other viruses that can be used, please see the review by Nayerossadat.

Adenovirus vs lentivirus

Can we pit a HIV virus loaded with CRISPR to remove the pathogenic HIV virus people are infected with? Sure we can. HIV virus belongs to family of lentiviruses. The cool thing about them is that they are good at staying in body for long term plus they make their genome a part of ours. So you get a cell intrinsic gene, within your own cells to do the job. However, the trick is to make this virus non infectious. Usually what we do is that we cut the genome of the virus into three parts. One part contains the viral genome along with your gene of interest. This part incorporated into new virus particles. But it lacks genes to make new virus particles or replicate. We put those genes on two seprate plasmids. Then, in a cell culture dish we put all of them together inside a cell line(usually HEK cells). We harvest the incompetent viruses made by these cells - that is these virus particles can insert genes into new cells, but can't make copies of itself anymore. These viruses can hence be safely used to deliver genes to live animals. By choosing the right envelope protein of virus particles you can choose the target cell of your choice(read my blog about making virus particles here).

Though, lentiviruses are very efficient and do their job very well, some safety concerns remain. For instance since you are integrating your genes inside human genome a wrong insertion site may lead to detrimental health affects. Plus, it is hard to predict long terms affects of having a foreign gene in your cells. But what if we don't insert the gene into the genome, we just had to express it transiently? Well, for applications of CRISPR-Cas9 which meant to just cut the bad gene or insert a new gene can do with it. And, Adenovirus will be perfect for our job.

Well, given the above information it should come as no surprise that most of the studies mentioned in my previous blog on CRISPR, used Adenovirus to deliver Crispr-Cas9 genes to live animals. Just like we did for lentivirus, making replication incompetent virus particles for adenovirus is not much different. The only difference is that they will deliver their genome into the cytoplasm of the cell, express the gene and then degrade. Moreover, they have an added benefit, that they deliver larger gene fragments to cells than lentivirus can₆.

To add to good news the clinical trial₇ in which adenovirus associated particles are used to modify cytotoxic T-cells is already on its way. Though in this trial the T cells will be modified outside the body of the patient and then inserted back(yeah just like those CAR-T cells you heard about). These modified T-cells are expected to treat cancers caused by EBV virus.

Nevertheless, the day is not far when we will soon be trying modifying the genome in vivo of humans. Though even for adenovirus the safety concerns remains. For instance, we will need to figure the right dose of virus to infect minimum number of target cells. The catch is to not cause a hyper immune response or any other side effect for the given dose.

Alright then, I guess now you are equipped with some basic amount of information for what all transporters exist, and what are being researched on. My favorite any day are viral transporters. However, we are still trying to optimise the transporters to get the maximum efficacy and safety. In fact, another clinical trial which will test CRISPR in removing HPV virus should soon begin. So, let's wait and watch. However, one thing I can tell you from our discussion is there won't be one transport we will be using. Our transport will be application specific.

References

1. How Cationic Lipid Mediated Transfection Works.

2. Son KK, Tkach D, Hall KJ. Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA. Biochim Biophys Acta. 2000 Sep 29;1468(1-2):6-10. PubMed PMID: 11018645.

3. Simões S, Filipe A, Faneca H, Mano M, Penacho N, Düzgünes N, de Lima MP. Cationic liposomes for gene delivery. Expert Opin Drug Deliv. 2005 Mar;2(2):237-54. Review. PubMed PMID: 16296751.

4. Kaido Kurrikoff, Kadi-Liis Veiman, Kadri Künnapuu, Elin Madli Peets, Tõnis Lehto, Ly Pärnaste, Piret Arukuusk & Ülo Langel, Effective in vivo gene delivery with reduced toxicity, achieved by charge and fatty acid -modified cell penetrating peptide, Scientific Reports, 2017

5. Microinjection

6. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery. Adv Biomed Res. 2012;1:27.

7. PD-1 Knockout EBV-CTLs for Advanced Stage Epstein-Barr Virus (EBV) Associated Malignancies

8. Study of Molecular-targeted Therapy Using Zinc Finger Nuclease in Cervical Precancerous Lesions

9. The secret Jedi life of the Virus - HIV as screwdriver for manipulating genes.

10. Journey from adaptive immunity in bacteria(CRISPR) to saving Man's best friend(Dog).

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I hope you enjoyed the article.
Signing off
@scienceblocks

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