A journey through the world of DNA delivery systems

by Mirco Martino

The field of medicine, this year, has been marked by the award of the Nobel Prize for Chemistry to Emmanuelle Charpentier and Jennifer Doudna ‘for the development of a method for genome editing”, the CRISPR/Cas9 system.

 Such incredible and powerful technology has brought genome editing into the spotlights. Now it is possible to modify a specific genetic sequence in a living organism to substitute a defect gene. This has been a dream of scientists since the discovery of genetics and now it is a reality.

 To make gene therapy available to everyone, there are still 2 hurdles: delivery and accuracy. Delivery of genetic cargo through the body barriers and the cells, to reach the nuclei, avoiding any immunological response of the organism is a significant challenge. Once inside the nucleus, the problems don’t disappear, properly finding and modifying the target sequence in live cells along the 2km of compacted DNA without any off-targets issues or insertion in unwanted places.

 Recent discoveries and technologies like CRISPR have provided turbo charged solutions to some of the problems however, we are still far away from perfect delivery system.

 In the majority of gene therapies in the current clinical trials or under development, the nucleic acid cargo  consist mainly of long DNA molecules but can be present in a variety of forms. Firstly there are plasmids containing the modified target sequence, then there are episomal replication features and original bacterial sequences. Finally, there are donor vectors for the homologous recombination trigger by CRISPR that included homologous arms and the sequence to incorporate in the genome, or other DNA-based sequences such as transposons system.

Due to their size and negative charge, it’s almost impossible to have an efficient delivery of such molecules without incorporating them into a vector.

The vectors can be divided in 2 main categories: viral and non-viral.

If the first group uses the natural system of viruses to deliver DNA to cells upon infection, the second includes all artificial molecules created to encapsulate the DNA molecules to be able to efficiently pass through the cell membrane.

 The choice of the delivery system should always be carefully matched to the intended recipient. For example, methods that nicely work for example in ex-vivo procedure could be ineffective or not feasible for a full body somministration.

Delivery systems can then be further sub-categorised as follows:

●     Physical delivery methods that, as the name indicates, consider the target system as a physical model more than a biological one: microinjection, electroporation, hydrodynamic;

●     Viral vector delivery methods that  take advantage of the natural infection system of the virus: adenovirus, adeno-associated virus and lentivirus;

●     Non-viral vector delivery system that uses the biological vesicles import system of the cells to absorb nanoparticles of different nature: liposomes and polyplexes.

Physical delivery methods:

Physical delivery methods include all systems in which physical properties and principles are used to force the entrance of the cargo in the target cells. Although they have a high transfection efficiency, they use complicated setup and instruments that make them costly and difficult to use as high-throughput techniques for clinical settings. Moreover, for now, they cannot be used for clinical trials if not for specific ex-vivo treatments where specific cells from the patient are collected for in vitro manipulation and after reintroduced in the patient.

One of the common advantages of the physical delivery methods is that they are not limited by the size of the molecules and complexes to deliver. The most used physical delivery methods include: microinjection, electroporation and hydrodynamics.

Microinjection:

With the use of 0.5-5µm needles and a microscope, by piercing the cellular membranes, the cargoes are directly inserted in the desired site of the cells. The advantage of this method, as expected, is that the amount and the site of the delivery is precisely known and controlled by the operator. The downside is that the process is slow, tedious and laborious as the molecules need to be delivered cell by cell and not all of them are able to survive after the injection. At clinical level it is used for ex-vivo gene therapies and for in-vitro fertilization.

Electroporation:

Electroporation is a well-established method that allows molecules with hydrodynamic diameter of around 10nm to enter transient nanometers-size pores created in the cell membrane by the application of high-voltage electrical current. As opposed to microinjection, electroporation is a good technique to use for cell population, it has quite high transfection efficiency and it’s quite easy to use in any standard research laboratory. The main advantage of electroporation compared to other delivery techniques is that it works in hard to transfect and nondividing cells. Some drawbacks are the need for heavy optimization to establish the correct operation conditions to use, the negative effects on the cell viability and that it is impossible to control the amount of cargo that will enter each cell. It’s also important to note that due to the use of high-voltage current, this methodology is only used for in-vitro or ex-vivo therapies.

However, instrumentation is improving and there are now products on the market with presets for delivering cargo to the nucleus of different cell types.

Hydrodynamics:

This is probably the technique that better represents the idea of physical delivery methods, since it uses the physical property of the blood as liquid, to deliver the desired cargo in-vivo.

Blood is an incompressible liquid, so, a large volume solution (10% of the body weight) with the cargo pushed in the bloodstream of the animal will cause an increase of the hydrodynamic pressure that will temporarily increase the permeability of the parenchymal and endothelial cells allowing the cargo to enter. The technique is limited in the organs that can be targeted, mainly the liver, kidneys, muscle and heart. Moreover, the transfection efficiency is quite low. Nevertheless, it doesn’t require any external vector or component and it’s easy to apply. Hydrodynamics could also be used in clinical trials but this hasn’t been possible yet due to physiological complications that can arise  including liver expansion, cardiac dysfunction and elevated blood pressure.

Viral vectors delivery systems:

One of the most genuine biotechnological applications is the use of the viral infection principles as delivery systems. Delivery systems based on viral vectors exploit one of the oldest biological replication systems as a technology to introduce cargo inside cells with high precision and efficiency. Viruses are able  to recognize specific cell types, let their genetic content enter  and have reproduced taking advantage of the replication machinery of the  infected cells. Viruses are divided in different categories according to the type of their genetic material, DNA (single or double strands) or RNA, and the type of capsid and membrane structures they are made of. Viral vector delivery systems use the original recognition and infection system of the specific virus family, but without all the components that would allow the viruses to duplicate and propagate causing a real infection. In these vectors, the desired cargo is inserted as genomic material of the virus itself. Viral vectors have many advantages: they can be designed to recognize a particular type of cell, they have a very high transfection efficiency, they protect the cargo until it is delivered to the cell andthey can easily be produced and utilized as delivery systems in a control setting. Moreover, specific viral vectors can be used to integrate the cargo in the host genetic material. At the same time, viral vectors are  probably the most dangerous system for clinical application if not well designed: they can integrate the cargo in undesired regions of the host genome, can infect the wrong cell types and, in the worst cases, can gain back the faculty to duplicate and infect the host and they can cause an immune response. Another limit of the viral vectors is the size of cargo they can deliver . Nevertheless, viral vectors are currently used successfully in some clinical applications.

The 3 main family of viruses used are: adeno-associated virus (AAV), lentivirus (LV) and adenovirus (AV).

Adeno-associated virus

AAV are probably the safest category of viral vectors: they don’t trigger an immune response and they are not known to cause or be related to any human disease. Moreover, in nature there aredifferent serotypes of AAV that can be used to target many specific cell types. A peculiar feature of AAV is that the genomic content can persist indefinitely in the cells as both exogenous DNA or, applying some modification, as integrated material in the host genome. That can be an advantage if we wish to keep a stable amount of our cargo in  the target's cells, or a disadvantage if the cargo needs to be removed. One limit of the AVV is that the size of genetic material they can deliver is limited to 4,5Kb.

Lentivirus

LV are RNA-type viruses that can infect both dividing and not dividing cells. Their main advantage is that it is possible to replace the viral glycoproteins with different ones that recognize a specific cell type, making it possible to use these vectors to target almost all types of cells. LV integrated the cargo in the host genome. As for the AAV, this can be an advantage or a disadvantage depending on the application. The integration site cannot be completely controlled and that can cause undesired deregulation of the host’s genes. Moreover, LV can activate an immune response. Generation systems to produce LVs are complicated since the different components of the virus have to be placed in separate plasmids to avoid that viable virus particles can accidentally be produced within the cells.

Adenovirus

As LV, AV can infect dividing and nondividing cells, including also quiescent and tumor cells. Differently from LV, they are DNA-based viruses of which genetic content doesn’t integrate but remain as an episomal vector in the host, preventing insertional mutagenesis. AV can also be modified to be able to replicate and lyse only  tumor cells, leaving the normal cells intact. The main disadvantage of AV is the strong immune response that they can trigger, that sometimes can be also lethal for the infected organism. This feature is used, anyhow, as an advantage in some clinical applications to recruit for example the immunity system to cancer cells or in some vaccination systems.

Non-viral vector delivery systems:

Nucleic acid cargo is generally quite unstable outside the cells, especially anionic nucleotides that don’t pass easily through the negatively-charged cell membrane. To resolve this issue, the cargo can be encapsulated in structures with a  neutral or a cationic charge. Those structures are delivered into the cells by endocytosis mechanism so the molecules inside are released within the cell cytoplasm. The main advantage of those methods are the safety, the easy procedure to create and use them and the low-cost production. Nevertheless, the transfection efficiencies are really cell type dependent and they cannot be cell-type specific. They are mainly used for in-vitro or ex-vivo applications. The major issues related to the endocytosis mechanisms used by the non-viral vectors to enter the cells are connected to the escape from the lysosome system and the transportation from the cytoplasm to the nuclei.

The main category of non-viral delivery systems are: liposomes and polyplexes.

Liposomes

Liposomes are cationic lipid molecules that when mixed with nucleotides form lipoplexes, structures that surround the cargo and protect it from internal and external cellular nucleases.  They are composed of cationic hydrophobic tails and anionic hydrophilic heads connected each-others by linkers. Neutral lipids and cholesterols molecules are often added as well to facilitate the escape from the endosomes. Lipids with different chemical properties can be used to form lipoplexes with specific features. Lipoplexes have been used  in-vivo application as well, but they have shown high toxicity and the ability to trigger the immunity system. Surface coatings are under study to reduce such side effects.

Polyplexes

Polyplexes are polyethenimine (PEI), poly(L-lysine) (PLL) or polyethylene glycol (PEG)  based structures with high-charge density and pH-buffering ability that facilitated the packing of the cargo and the escape from the endosome. They have a limited toxicity and new polymers are under study to increase transfection efficiency and reduce the toxicity of polyplexes.

Final Remarks

Those listed above are only a few of the existing delivery methods. Many others exist but haven’t been extensively studied. Researchers are trying to take advantage of different biological or physical properties to increase the transfection efficiency and reduce the unwanted effects and the toxicity.

For clinical applications, the most used delivery systems are probably the AAV-based vectors although their size limits a lot of possible applications, especially with long DNA molecules. New formulation of liposomes and polyplexes can in the future be a valid alternative to deliver long DNA nucleotides also in clinical applications where local injection is possible. For now, the perfect delivery system doesn’t exist and many factors, including type of transfection (in-vitro, ex-vivo, in-vivo), type of cells, cost and toxic effect, has to be considered when a delivery system needs to be chosen.

Moligo Technologies provides long and ultra-pure single stranded DNA oligos at industrial scale. Single stranded DNA is often easier to deliver to cells, for more information about our products and our methods, book a call with someone in our technical team.

References

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●     Wang, D. & Gao, G. State-of-the-art human gene therapy: part I. Gene delivery technologies. Discov Med 18, 67–77 (2014).

●     Slivac, I., Guay, D., Mangion, M., Champeil, J. & Gaillet, B. Non-viral nucleic acid delivery methods. Expert Opin Biol Th17, 105–118 (2016).

●        Li, S. & Ma, Z. Nonviral Gene Therapy. Curr Gene Ther 1, 201–226 (2001).

●        Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology 208, 44–53 (2015).