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As gene therapies expand their clinical development and therapeutic reach Biotech’s are always looking for better ways of tackling the biggest hurdle in gene therapy – the safe and efficient delivery of the genetic material to patients. In this blog post we look at the pros and cons of delivery via viral Vs non-viral vectors.
Why All the Attention on Vectors?
Gene therapies have gained much attention in recent years, with their transformative potential recognized by investors and developers alike. The rise of gene therapies has created a debate – on whether to use viral or non-viral vectors to deliver this genetic material to patient cells.
Designers, developers, and manufacturers choose which of these two vector variations to integrate into gene therapy production based on three main factors:
- Vector safety
- Target efficiency
- Feasibility
According to Global Data there are currently 4 approved gene therapies and 3 gene therapy products in the pre-registration stages.
15 products are currently under Phase III investigation for a range of indications including hematological disorders, ophthalmology, immunology, and infectious diseases, and, a further 277 gene therapies in Phase II clinical trials.
With the evidential clinical success of gene therapies growing so does the interest in vectors. So which should you be using and why?
Viral Vectors
Viral vectors, engineered from the blueprint of a virus, capitalize on the virus’s ability to enter the nucleus of a cell and deliver genetic material.
The choice of viral vector depends on many factors depending on the needs of the disease to be targeted, including the amount of genetic material to be delivered, properties and location of the cells being targeted, and safety considerations – limiting the possibility of an immune response.
General Pros of Viral Vectors:
- Improved safety – The production of viral vectors only incorporates the aspects of the virus genome which make it good at packaging and delivery. The parts coding for the viral infection are left out of the vector make-up, rendering the virus non-pathogenic.
- Transduction efficiency – Viral vectors are highly efficient at transducing genetic cargo into host cells, delivering the full intended therapeutic effect of the gene therapy.
- Decades of development – Viral vectors have been thoroughly investigated over the last few decades consequently scientists are well versed in every aspect of them. Their use in gene therapies has presented positive clinical outcomes targeting many diseases, including, cancer, infectious diseases and monogenic diseases.
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General Cons of Viral Vectors:
- Immune response- Despite not carrying the ability to infect the host with the virus’s original infectious material, the foreign nature of viral vectors can elicit an immune response – both innate and adaptive. This can prevent the long-term use of viral vectors and the efficiency of transduction of delivered genetic material.
Understanding how the immune system interacts with viral vectors and controlling these responses would extend their clinical use. Approaches currently include using novel vectors with tissue-specific tropism, using alternative routes of administration and using genetically engineered capsids.
- Manufacturing – Viral vectors can present complex challenges in manufacturing processes contributing to high production costs and difficulty when manufacturing on a commercial scale.
Efforts to reduce manufacturing complexities and costs of viral vectors have included: using stable producer cell lines rather than transfected producer cells, stereotype-independent processes, and, the use of suspension rather than adherent cell culture.
- Reliance on animal models – Testing viral vectors in animal models can cause inaccuracy in predicting the transduction.
Using humanized mouse models or tissue grafts on animal models can be used for greater accuracy when considering the efficacy of viral vectors in humans.
There are 3 main types of viral vectors: Adeno-associated viral, lentiviral and retroviral – all with their own pros and cons.
Adeno-Associated Viral Vectors
The most popular viral vector used for in vivo gene therapy is the adeno-associated viral (AAV) vector. A non-pathogenic parvovirus, with 11 stereotypes and more than 100 variants each with a different tropism, AAV’s can specifically target many different tissue types.
They have previously been used in gene therapies targeting: ophthalmological diseases, metabolic diseases, hematological diseases, neurological diseases, and musculoskeletal diseases.
Pros and Cons:
- Adeno-associated viral vectors can carry and deliver multiple genome copies into one host cell for higher gene expression.
- These vectors deliver genetic material without merging it into cell DNA, therefore the new genetic material cannot be passed down to daughter cells keeping the genetic expression transient.
- They can transduce both non-dividing and dividing cells allowing for long-term stable gene expression.
- AAV’s are very stable with low immunogenicity.
- As the genetic material is not inserted into the host genome, therefore in dividing cells the daughter cells do not express the added gene and the therapeutic action is lost.
Retroviral Vectors
An enveloped spherical virus, retroviruses carry genetic material in the form of RNA, reverse transcribing this RNA into DNA before integrating it into the host cell genome. However, retroviruses are no longer selected for use in new clinical trials.
Pros and Cons:
- Retroviral vectors can carry a large amount of genetic material – up to 12kb.
- The resultant gene from reverse transcription of the RNA is integrated into the host genome resulting in long-term gene expression.
- Retroviruses can only deliver gene therapies directed at dividing cells as they need the host cell to divide in order to integrate DNA into its genome.
- The is a risk of random insertion of the vector DNA into the genome causing insertional mutagenesis. Retroviral vectors that have the DNA’s promoter or enhancer long terminal repeats programmed to delete can reduce this risk.
Lentiviral Vectors
Lentiviruses are a subtype of the retrovirus and similarly carry its genetic material as RNA, reverse transcribed into double-stranded DNA before transduction into the host genome.
These vectors are mainly used in ex vivo gene therapies but also show promise in clinical trials for their application in vivo, for monogenic and chronic diseases such as neurological, ophthalmological and metabolic diseases.
Pros and Cons:
- These vectors deliver the genetic material directly into the DNA of cells. Genetic changes are passed onto daughter cells resulting in long-term gene expression.
- Unlike Retroviruses, lentiviruses can integrate into and transduce both non-dividing and dividing cells.
- Modern generations of lentiviral vectors are derived from non-human lentiviruses, rather than the human immunodeficiency virus 1 they were originally developed from. This makes them viewed to be safer and preferable as the parent virus is not infectious to humans.
- They have a lower risk of genotoxicity and insertional mutagenesis than retrovirus vectors.
- Lentivirus vectors have a limited capacity for carrying genetic material.
Non-Viral Vectors
Whilst viral vectors continue to be used in approved gene therapies and most recent clinical studies, their high manufacturing expenses and difficulty to translate to a commercial scale means research to find alternatives has gained pace.
The most notable non-viral vectors include polymers, lipids, peptides, inorganic particles, or, hybrid systems.
General Pros of Non-Viral Vectors
- Commercial scalability – Generally non-viral vectors are more easily manufactured than viral vectors. This improves scalability and reduces costs, placing gene therapies in a better commercial position.
- Better safety – Non-viral vectors cause low levels of cytotoxicity, immunogenicity and insertional mutagenesis. Having a robust safety profile reduces the risk of side effects to patients receiving gene therapies and also helps to improve public opinion towards these advanced therapies.
- Large cargo capacity – They can also provide a larger cargo packing capacity than most viral vectors, reducing the amount of product needed for the intended therapeutic effect, increasing manufacturing and administration efficiency.
General Cons of Non-Viral Vectors
- Poor gene transfer efficiency – Non-viral vectors must find a way to protect its genetic material from endosomal degradation during transfection. There is a greater risk of destruction and consequently lower levels of new gene expression. These vectors often require customized systems to protect them.
- Low levels of specificity – It is a challenge to ensure that non-viral vectors deliver their genetic cargo to the intended tissue type. A lack of specificity can be harmful resulting in toxicity, and unexpected side effects, alongside the lack of therapeutic effect.
- Make-up Complexity – Non-viral vectors often utilize hybrid systems that can make for very complicated vector formulations. For example, the Pfizer/BioNTech vaccine used a vector composed of 4 lipids. With so many different hybrid combinations to choose from screening for a successful vector can take a very long time and predicting how the vector will behave adds complexity.
Let’s look at the specific pros and cons for the non-viral vectors dominating this area of research.
Polymers
Cationic polymers, such as polyethylenimine (PEI), hold a versatile chemical structure with a high capacity for holding genetic material, which they can neutralize to form a polyplex that is then easily transported.
Pros and Cons:
- PEI has the greatest transfection efficiency of any non-viral vector. The positive charge of cationic polymers has multiple benefits, including generating an osmotic effect to induce endosome burst and assist transfection efficiency.
- PEI’s are also able to escape endosomal vesicles when they deliver the genetic material across the intracellular membrane.
- However, PEI is non-biodegradable and accumulates around cells causing cytotoxicity.
- A lack of cell delivery specificity and transfection has been observed in PEI vectors.
- Synthetic polymers that are biodegradable are under investigation to addresses the issues of cytotoxicity, such as PBAEs and PLAs.
Lipids:
The properties of lipids have long made them a clear non-viral vector choice to deliver genetic material to cells. The positively charged groups in most lipids form electrostatic interactions with the negatively charged genetic material to be carried creating lipoplexes.
The first siRNA gene therapy Onpattro to be FDA approved uses a lipid-based vector.
Pros and Cons:
- Lipids are biodegradable, therefore the risk of cytotoxicity is reduced.
- Lipids with ammonium head-groups make good gene carriers due to their positive charges. However, these charges can cause toxicity and the lipids have a short half-life limiting their applications in vivo.
- Modified lipids have been developed that have positive charges to form lipoplexes with DNA and escape endomsomal vesicles, but that also have a neutral charge at the physiological pH to allow for delivery.
- Butanoate is the lipid vesicle used in then FDA-approved Onpattro and has excellent gene silencing activity. It’s derivative CD-Chol is used commercially in cancer gene therapy clinical trials.
Inorganic Materials
Due to their stability, inorganic materials are a non-viral vector of interest. The inorganic material to demonstrate the best carrier abilities are silica-based systems such as silica-nanoparticles, gold-nanoparticles, magnetic nanoparticles, and carbon nanotubes.
Pros and Cons:
- In-organic materials are more stable than organic materials.
- Gold nanoparticles are less cytotoxic in comparison to alternative in-organic vectors.
- Carbon nanotubes possess properties to facilitate transfection independent of the endocytosis process for efficient transfection.
So Where Does the Future of Vectors Lie?
As with many areas of science, the answer lies in more research.
If we are to see viral vectors replaced by non-viral vectors for common use to deliver gene therapies we need to know more about how they can improve upon the already well established viral vectors in common use today.
Both viral and non-viral vectors hold characteristics favourable for vector delivery. If the issues regarding inefficient transfection are resolved in non-viral vectors then the benefits they offer in scalability, lower manufacturing costs and greater flexibility could offer a desirable alternative to popular viral vectors such as AAV.
References
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