CRISPR/Cas9 delivery: A New Technology Faces an Old Problem
posted February 6, 2017
Since its revolutionary introduction, you would be hard-pressed to find a scientist that has not heard of CRISPR/Cas9 gene editing technology. CRISPRs, or clustered regularly interspaced short palindromic repeats, are derived from the immune system of bacteria, where they have been used for eons as a defense mechanism against invading viruses. When CRISPRs are transcribed into RNA, they will specifically look for a certain target sequence found on strands of DNA, encoding the cutting enzyme Cas9, and cut the DNA at that location. In bacteria, this strategy means that the CRISPRs can target the DNA of viruses, cutting the strands and rendering the virus useless. For human researchers, it means we can use this highly customizable system to target and modify any part of the genome in a cut-and-paste manner, potentially allowing us to fix the DNA mutations that cause a myriad of currently incurable diseases.
Since the implementation of CRISPR/Cas9 engineering, its impact has been evident in the amazing progress in gene editing. This is particularly true in the field of muscular dystrophy, where the mutation causing this disease has been successfully edited out in a mouse model. However, CRISPR technology is hampered by something that has been a long-standing problem in other fields such as cancer research: the delivery of CRISPR into the target cell type.
The successful delivery of any therapeutic, such as cancer drugs, is faced with many challenges. The drugs must stay stable when injected into the blood stream until they reach the tissue, but since the drugs are toxic, precautions must be taken to ensure they do not enter any off-target tissues like the liver, causing detrimental side effects. Similar to cancer drugs, it is undesirable to have CRISPR/Cas9 degrade before reaching its target cells. Also, since CRISPR is such a powerful genome editing tool, we don’t want it to enter the wrong cell type where it might begin cutting off-target DNA. If that isn’t enough, unlike drug delivery, CRISPR/Cas9 has the added challenge of delivering both an RNA sequence (CRISPR) and a protein (Cas9), which have differing chemical and physical properties.
To Viral Vector or not to Viral Vector
Currently, scientists researching drug delivery use a variety of strategies to meet the above criteria. Drugs can be placed in protective nanoparticles made up of water-insoluble lipids that can transport them safely through the circulatory system. They can also be tagged with ligands (i.e., proteins) that will target and cause entry of the drug only into a specific type of cell; for example in breast cancer therapy many delivery systems make use of HER-2, a protein receptor that is overexpressed only on the surface of breast cancer cells. Researchers are currently exploring two main methods for CRISPR/Cas9 delivery: viral and non-viral vectors. Viral vectors, such as adeno-associated shuttles, use the natural shell of viruses as a cargo bay. They have a high efficiency of delivery and low immunogenicity, but suffer from safety concerns and cannot carry very much material. Some researchers have looked into methods to increase their packing capacity, including delivering CRISPR and Cas9 in separate vectors, and using a smaller molecular weight version of Cas9. Non-viral vectors, like those mentioned for cancer drug delivery, make use of artificial lipid- or polymer-based nanoparticles to transport the CRISPR/Cas9 system. Due to the negative charge of RNA, it can be encapsulated in positively charged particles simply by using electrostatic interactions. This strategy avoids the potential safety concerns with the use of a viral vector, allows for the large-scale synthesis of carriers, and the physical and chemical properties of the carrier can be tailored to suit each delivery need. Although it might seem like nanocarriers are the superior system, they are often associated with low transfection efficiency, poor stability, and immunogenicity. It is clear that as the variety of therapeutic targets continues to expand, researchers employing CRISPR will need to turn their focus to the delivery method. While viral vectors are a more established and well known strategy for gene delivery, researchers may need to employ synthetic polymers in order to match the customizability of the CRISPR technology and translate it into clinical practice.