Section III: Advanced Drug Delivery Topic - Vectors for gene/drug delivery

Paper reviewed - Pezzoli, D., Chiesa, R., De Nardo, L., & Candiani, G. (2012). We still have a long way to go to effectively deliver genes! Journal of Applied Biomaterial and Functional Materials. (10). 82-91.

Before rare diseases are widely known, researchers focused on developing drugs that are highly demanded because the same effort could help more patients. The progress of bioinformatics has made gene sequencing more accessible and scientist begin to find out that most rare disease are related to the gene, mostly inherited or mutation. Compared to 40 years ago, the survival rate or cancer has increased 40% upon diagnosis by killing abnormal cells using chemotherapy, immunotherapy, etc. However, due to the rapid spreading and spontaneous mutation, it is extremely difficult to kill abnormal cells completely. Also, patients do not have the same response to cancer drugs. Currently, there are no positive data that shows desired efficiency of novel drugs that treat gene mutations. This review mainly covers gene therapy with viral and biodegradable polymer.

Examples of inherited or non-inherited gene mutation are cancer, HIV infection, cystic fibrosis, haemophilia, diabetes, severe combined immune deficiency, etc. They can be restored by selectively introducing therapeutic genes into target cells – known as gene therapy. The negatively charged cell membrane and DNA produces electrostatic repulsion, hence, a viral or non-viral vector is required to insert gene into the cell. To date, gene therapy products are only approved for veterinary uses. For example, DNA Vaccine (Norvatis Animal Health) that uses Alphavirus to treat infectious salmon anaemia. Gene therapy is categorised into somatic cell and germ line gene therapy (Table 1). Also, according to patients’ situation, introduction of therapeutic genes includes DNA, siRNA, CRISPR, shRNA, mRNA, etc (Table 2).

Researchers have been intensively looking for an efficient and safe vector to deliver genes into problematic cells. One of the most efficient, but toxic carrier is the virus. First, viruses encapsulate genes within the envelop or capsid that protects gene from enzyme degradation; secondly, it has replicative gene that allows rapid reproduction of gene in the body, which makes it as an efficient gene carrier. However, the it contains the virus gene that would cause HGT into host genome, causing harmful oncogenic mutation. Therefore, viruses are engineered to have its toxic gene removed, so that only therapeutic genes are introduced into the body.

In a recent review, Srivastava & Carter (2017) opposed Nault, that AAV-2 is not associated to Hepatocellular Carcinomas (HCC) because there was only 1% of the Korean population with HCC, was infected by AAV2. The review also stated that AAV2 selectively induces apoptosis on cells that has p53 malfunction; where p53 is responsible on preventing cancer formation. However, there was a manipulated factor that the oncogenic AAV2 may be aged related, that older population is more likely to be infected (Figure 1).

Non-viral gene therapy includes electroporation, naked DNA, molecular conjugates, gene gun and polymer. It has relatively poor transfection efficiency compared to viral vectors, some even possess equivalent toxicity as viral vector does. Many polymers with variety of structure, shape, and characteristics were developed for gene therapy, but only a few made it to clinical trials.

Polyethylenimine (PEI) comes into one’s mind during discussion of non-viral polymeric gene vector. It has the best transfection efficiency due to the presence of high percentage protonated amine groups. Gao et al (2016) found that the transfection efficiency of a polymer is proportional to its molecular weight (MW) - 25kDa is more efficient than 8kDa PEI. Also, Godbey (1999) stated that size affects transfection efficiency, the same polymer with a higher MW has higher cellular uptake. However, it is not biodegradable upon transfection, where gene is not released completely and induces cytotoxicity.

Biodegradable polymers are seen to be potential vectors that are safe since it degrades in the cell. Examples include poly(beta-amino esters) (PAE), polymers with disulphide or diselenide backbone, etc. Transfection efficiency can be improved by either increasing the chain length, or incorporating these structures into non-biodegradable polymers with low MW. For example, conjugating low MW PEI to the biodegradable backbone polyglutamic acids, has significantly lower cytotoxicity and higher transfection efficiency compared to PEI-25kDa (Wen et al, 2009). PAE polymers can be synthesised through simple one-pot Michael addition and are cleaved in the presence of water molecules.

Figure 2. Molecular structures of biodegradable polymers.

Godwin et al (1991). Drug resistance tumour cells are related to the drastic increase of glutathione (GSH) concentration in the cell, 13 to 15 folds higher than drug sensitive cells. GSH is responsible to promote proliferation and inhibit cell death by regulating carcinogenic mechanism, sensitivity against cytotoxic drugs, ionising radiation (Godwin et al, 1991; Ortega et al, 2011). Polymers with disulphides are outstanding as it undergoes redox reaction with GSH, disintegrate and release gene or drugs into the cell, while the oxidised GSH could not affect the cytotoxic drug/gene. On the other hand, if polyplex is transfected into a healthy cell, the GSH level will be too low to initiate gene release, promoting selectivity of gene therapy. Compared to PAE, disulphide bonds are sensitive to oxygen, light and temperature; therefore, the synthesis of biodegradable polymer with disulphide backbone is way more complicated.

Cancer cells mutate extremely fast and different people have different response on the same drug. Being aware of the risk of drug-drug interaction, there is a trend of using two or three drugs in combination for better therapeutic effects. In my opinion, more research should be done on bioinformatics, finding out which drugs are more beneficial to the patient; or which molecules are more readily taken up by cells by fitting into the receptor and incorporate such molecules on the vector. One could not define whether viral or non-viral is a better vector because even if gene is transfected into the cell safely, another problem arises because the possibility of HGT is very high – causing more mutations.

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