technology review: gene editing platforms - phacilitate.co.uk · cell types. simple process...

Technology Review: Gene Editing Platforms

In partnership with

Technology Review Series: Gene Editing Platforms

2www.phacilitate.co.uk

Non-viral platformsNon-viral approaches to cell engineering leverage membrane-disruption or carrier-mediated techniques to introduce foreign material into a cell, with the intention of modifying its function. The two main physical, non-viral modalities are membrane-disruption, including permeabilising the membrane through thermal, electronic, hydrostatic or other means; or physically penetrating the membrane, such as with a nanoneedle. Carrier-mediated approaches leverage endocytosis or fusion-mediated methods to delivering payloads, typically using bio-inspired platforms such as exosomes, vesicles, or lipid conjugates, while various nanotechnology approaches are also subject to research.

One major advantage of non-viral approaches is that a range of different materials can be inserted into the cell. Viral approaches rely on a vector to deliver a nucleic acid, which in turn requires transcription and/or translation to give protein functionality. Not having to manufacture viral vectors is another upside to these approaches, as this manufacturing step is complex, costly and is a limiting factor when scaling up overall production of the commercial, therapeutic product.

Non-viral approaches can also deliver alternative nucleic acids such as siRNA or miRNA, in addition to peptides, small molecules, nanomaterials, DNA origami, artifi cial chromosomes or gene editing constructs such as TALENs or CRISPR-Cas9. To date, no cell therapy using a non-viral gene-editing technology has been brought to market, despite some early clinical successes.

Various innovative techniques for non-viral cell engineering are described in research literature, and may become more clinically or commercially applied over the coming years. Meanwhile, there are several commercially available platforms used for preclinical and clinical development of cell therapies. Some of them are highlighted below.

Mechanical membrane permeabilisation SQZ Biotech

1 Sharei, A. et al. A vector-free microfl uidic platform for intracellular delivery. Proc. Natl. Acad. Sci. U. S. A. 110, 2082–7 (2013). 2 Sharei, A. et al. Cell squeezing as a robust, microfl uidic intracellular delivery platform. J. Vis. Exp. e50980 (2013). doi:10.3791/509803 Saung, M. T. et al. A Size-Selective Intracellular Delivery Platform. Small 12, 5873–5881 (2016).

Overview: Developed at the Harvard Stem Cell Institute in collaboration with MSKCC, the basis of the ‘CellSqueeze’ platform is to use a microfl uidics approach to disrupt the cell membrane by physically squeezing the cell through a construction channel 30-80% of the cell’s diameter. The shear forces resulting from such mechanical deformation form transient holes in the membrane that allow for the diffusion of material in the surrounding buffer into the cytosol. The platform has been shown to be capable of delivering a range of materials such as carbon nanotubes, proteins, and siRNA to 11 cell types, including embryonic stem cells and immune cells.1 SQZ Biotech are currently using the platform to develop antigen presenting cell (APC) cancer vaccines, in collaboration with Roche.

This is an exciting development in an area of high unmet need and CellSqueeze offers an intriguing solution with a highly credible scientifi c background. The existing collaboration with Roche represents good early promise, but the platform is yet to be validated in the fi eld of ex vivo gene therapy.

Advantages: Lower-risk and cheaper than viral approaches. The approach is moderately scalable, with one device shown to process millions of cells per second. Importantly, the technique is ‘gentle’ on cells and highly effi cient, showing a cell viability of 80% to >95% and transduction effi ciency of up to 75%, depending on the speed of cell processing.2

Disadvantages: There is a potential limitation to the correlation between cell size and delivery effi ciency. A microfl uidics channel of a fi xed diameter may not suffi ciently squeeze a small cell to disrupt the membrane, while larger cells may not even pass through the channel or may be structurally damaged. However, this issue can provide for size-selective cell transfection, which could be useful in some applications.3



Chemical membrane permeabilisation Avectas

4 O’Dea, S. et al. Vector-free intracellular delivery by reversible permeabilization. PLoS One 12, e0174779 (2017)5 Aijaz, A. et al. Biomanufacturing for clinically advanced cell therapies. Nat. Biomed. Eng. 2, 362–376 (2018).6 Li, L.-H. et al. Highly Effi cient, Large Volume Flow Electroporation. Technol. Cancer Res. Treat. 1, 341–349 (2002).

Overview: Avectas are developing a chemically-mediated platform that temporarily permeabilises the cell membrane through exposure to a proprietary solution containing a low concentration of ethanol and controlled de-permeabilisation. Cell permeabilisation can be reversed by selectively controlling cell exposure to the solution. Proteins, mRNA, plasmid DNA and other molecules have been delivered both alone and in co-delivery formats to a variety of cell types, including primary cells. Low toxicity and cargo functionality have been shown in proof-of-principle studies. The procedure lasts less than fi ve minutes, reporting a delivery effi ciency of 53% and cell survival of 78%.4 The company are reportedly developing a GMP-compliant closed-process confi guration.5

Advantages: Few and simple materials are used, reducing complexity and costs. Transduction and cell survival results are good in proof-of-concept studies and the company are now developing a commercial process. The platform is capable of transfecting several different cargo types.

Disadvantages: Proof of concept studies delivered the permeabilising solution to adherent cells using an atomiser sprayed onto a cell monolayer. Suspension cells were transfected by isolating cells in 12-well plate in a procedure that requires manual intervention. The approach may therefore require substantial development to be of a relevant scale for commercial development.

Electrical membrane permeabilisationMaxCyteOverview: Arguably the best-established transfection approach, electroporation has been widely used in both research and industry for many years. Electroporation involves disrupting the cell membrane by the passing of an electrical current across the cell, creating temporary holes in the membrane that are sustained as long as the current is maintained. MaxCyte was the fi rst company to report a large-volume industrial electroporation bioprocess, in 2002.6 Their current electroporation platform, Flow Electroporation™ Technology, now reports transfection effi ciencies of >90% using a range of payloads, and cell viability of >90% across the most commonly used cell types. MaxCyte have developed a closed, GMP-compliant process that has been used in the manufacture of some CAR-T products. The platform has also been used to transfect cell lines for large-scale production of lentiviral vectors.

MaxCyte’s electroporation technology is very well established, having been widely validated for clinical use in many therapeutic areas. Although an older technology, MaxCyte have optimised the electroporation approach to a high degree and provide a reliable and effective solution to cell engineering.

Advantages: The platform offers very good scalability, transfecting several million cells per second, and up to 200 billion cells in in one run that takes around 30 minutes. The platform has also reported some of the highest transfection rates as compared to other platforms.

Disadvantages: Electroporation has historically been known to cause some degree of damage to cells. The method is non-specifi c and allows molecules of any type under a certain size to transport in or out of the cell during the electroporation process.



LonzaOverview: Lonza’s electroporation transfection platform, 4D-NucleofectorTM Transfection Technology, was originally developed for research use but now has a larger-scale, closed-process, GMP-compliant version that has also seen clinical use. The platform uses the same fundamental electroporation principles that are shared with other such platforms. Lonza states 70% transfection rates with peripheral blood mononuclear cells and 90% for unstimulated human T-cells, with the ability to transfect one billion cells in a single batch.

Lonza have demonstrated suitability of the platform for transfection of DNA vectors, shRNA, mRNA, and siRNA oligonucleotides into hard-to-transfect cell types.

Advantages: Scalable and large-scale, high transfection rates for some cell types, relatively lower cost. Similar products available for research applications, supporting R&D/GMP process comparability. Simple process with low technical skill required.

Disadvantages: Lower cell survival and moderate transfection effi ciency in some cell types. Non-specifi c cell permeability allows movement of all molecules of a given size.

ThermoFisherOverview: The Neon Transfection System by ThermoFisher Scientifi c is an iteration on the electroporation approach that deploys microfl uidics to minimise the required voltage, reducing the risk of cell damage. Passing cells through a capillary of 0.56mm width and localising the electrical fi eld to a small area has the effect of reducing pH fl uctuations caused by the fi eld, which is thought to improve cell viability. The platform is shown to reach 90% transfection effi ciency in some cell types, with the ability to transfect from 2×104 to 6×106 cells per reaction. Specifi cally, peripheral blood mononuclear cells can be transfected with 23% effi ciency and 95% viability by using this technology.7

Advantages: Excellent cell viability, high throughput, and high transfection rates in some cell types. Simple process amenable to scale up.

Disadvantages: The system has reduced fl exibility in determining pulse parameters, both in terms of duration and voltage, leading to mixed transfection rates depending on application. Fine-tuning pulse parameters is a common approach to optimising the process for specifi c cell types and specifi c payloads. The platform also suffers from high cost of consumables.



Viral vector typesBelow we briefl y discuss each major type of virus class and compare their characteristics.

Viral platformsOverview: Viral approaches to cell engineering leverage the natural ability of viruses to deliver genetic material into the cell, which is then expressed as an episomal vector or integrated into the host genome for subsequent protein expression and phenotypic alteration. Viral ex vivo gene-editing methods have been successfully implemented in clinical trials and commercial products for decades.

There are four main groups of virus types in consideration for gene therapy: lentiviruses, adeno-associated virus (AAV), other types of retroviruses, and adenovirus. Retroviruses are integrating viruses and are more commonly used in ex vivo applications while adenovirus is non-integrating and usually used in vivo. Lentivirus and AAV are the two most commonly used types.

Advantages: Depending on the virus type used and the specifi c application, the approach benefi ts from a good track record of clinical success, high effi ciency and good specifi city. Some viral approaches to cell engineering have been validated as safe and effective solutions, as demonstrated by their widespread use in clinical development and commercial products.

Disadvantages: Viral cell engineering approaches do, however, have drawbacks as compared to non-viral alternatives. Viral vectors cannot deliver intracellular proteins and peptides, or indeed any payload other than genetic material, limiting their fl exibility. Further, the high cost, technical infrastructure and current signifi cant production bottleneck associated with vector production introduces a major cost and risk element to supply chains. Safety risks associated with the use of viral vectors do remain but present decreasing concern as developers learn more about how to manage these risks.

AdenovirusAdenovirus (AV) is a class of viruses largely responsible for respiratory infections, such as the common cold, pneumonia, and conjunctivitis. AV is non-integrating; it’s genome remains in the host cell nucleus and is expressed as an episome. Advantages of using an AV for therapeutic purposes are the ease of purifi cation and concentration, conferring an ability to deliver relatively higher titres, and the high effi ciency of transfecting both dividing and non-dividing cells regardless of the stage of cell cycle in a manner non-specifi c to the cell type. AVs can carry a payload of 7-8kb. Because the vector is episomal, it does not replicate when the cell divides, meaning it is diluted out of cell populations when expanded but also reduces the risk of insertional mutagenesis. Safety issues do remain however, and AV has been linked to some patient deaths. The effects of a vector delivered by AV are usually transient compared to those of an integrating virus. The characteristics of AV mean it is more commonly used for in vivo applications than ex vivo, such as in treatments for cystic fi brosis.

Adeno-associated virusAdeno-associated virus (AAV) is not known to cause any disease pathologies, and generally demonstrates much less immunogenicity than other viral vector types. AAV can however still illicit an immune response, complicated by the fact that most people have already been exposed to AAV, and have therefore have some pre-existing serotype-specifi c immunity. Wild type AAV is dependent upon co-infection alongside other viruses, predominantly adenovirus, in order to replicate. Although wild type AAV has been shown to integrate into the cell genome upon infection, recombinant AAV can be engineered to remain as a non-integrating episome, making it a useful gene therapy resource.



RetrovirusRetroviruses (RVs) are RNA-based viruses that integrate themselves into the host cell genome, allowing the virus to remain in every progeny of the infected cell. By changing the proteins present on the viral envelope, RVs can be directed to different cell types, but are generally unable to infect non-dividing cells. RVs can carry a relatively large payload of around 8kb, but are produced at lower vector titre, are diffi cult to concentrate, have lower transfection effi ciency, and are relatively instable. Their integration into the host genome can be diffi cult to specify and control (depending on the exact RV type), resulting in a risk of insertional mutagenesis that introduces safety and regulatory concerns. RVs have as a result generally fallen out of favour with modern cell engineering techniques, with exception of the lentivirus, which has become more widely used for reasons explored later.

Table 1:Key characteristics and qualities of four main viral vector types. MOI = multiplicity of infection.

Retrovirus Lentivirus Adenovirus AAV

Broad host range (infects many cell types)

Yes; dividing cells only Yes Yes Yes

Infects both non-dividing and dividing cells

No; dividing cells only Yes Yes Yes

Genome integration Yes Yes No No

Payload size (application dependent) 2.5–5.0 kb 2.5–5.0 kb 3.0–8.0 kb 2.5 kb

Typical titre 106 IFU/ml 107-108 IFU/ml 109 IFU/ml >1010 genome copies per ml

MOI rate ability Low (≤10) Low (≤10) High (>25) High (>25)

Expression pattern Stable Stable TransientStable;

site-specifi c integration

DrawbacksRisk of

insertional mutagenesis

Risk of insertional mutagenesis

Elicits strong immune response

Requires helper virus for activation; diffi cult to purify

LentivirusAlthough technically a genus of retrovirus, lentivirus is unique in that it is the only retrovirus that is capable of infecting both dividing and non-dividing cells. Lentiviruses include HIV and other causes of chronic diseases, often featuring long incubation periods. The natural ability of lentivirus to integrate to the genome of both dividing and non-dividing cells them appealing tools for therapeutic genetic engineering applications. Lentivirus also benefi ts from high transfection effi ciencies, stable long-term expression of the transgene, low immunogenicity, and the ability to accommodate relatively large transgenes. Platforms based on the lentivirus have therefore become arguably the single most widely used viral approach to gene editing, being used in various ex vivo gene therapy products (such as Strimvelis), as well as various CAR-T applications.



8 Stewart, M. P., Langer, R. & Jensen, K. F. Intracellular Delivery by Membrane Disruption: Mechanisms, Strategies, and Concepts. Chem. Rev. 118, 7409–7531 (2018).

Viral vector providersThe landscape of companies offering viral vector manufacturing services is growing signifi cantly, with some estimates predicting a market value of $51.3 million by 2022. There are a number of factors driving this growth, including the increasing focus on advanced therapies to treat an increasing prevalence of life-threatening diseases; increasing healthcare costs and growing global populations.

This table provides a summary of contract manufacturing and other organisations that are providing viral vector manufacturing services, with differentiation between viral vector types.

Table 2: Companies offering viral vectors for clinical or commercial supply AAV = Adeno-associated virus

Retrovirus Lentivirus Adenovirus AAV

4D Molecular Therapeutics ✔

Addgene ✔

ATVIO ✔ ✔ ✔

Batavia ✔ ✔ ✔ ✔

BioNTech ✔

BioReliance ✔ ✔ ✔ ✔

Biovian ✔

Brammer Bio ✔ ✔ ✔ ✔

Cobra Bio ✔ ✔ ✔

Eurogentec ✔ ✔

Finvector ✔ ✔ ✔

FujiFilm Diosynth ✔ ✔ ✔

Lentigen ✔ ✔ ✔

Lonza ✔ ✔ ✔

MassBiologics ✔ ✔ ✔

MolMed ✔ ✔

Novasep ✔ ✔ ✔ ✔

Oxford BioMedica ✔

ThermoFisher Scientifi c ✔

ViralGEN ✔

VIVEbiotech ✔

Vivegene ✔ ✔ ✔

Waisman ✔ ✔ ✔

WuXi AppTec ✔ ✔ ✔ ✔

Yposkesi ✔ ✔

Starting materialWhether a gene editing platform is viral or non-viral, plasmid DNA represents a critical starting material for the process. One to four plasmids are in fact necessary to start a non-viral procedure (e.g. transposon) or to manufacture viral vectors. Companies active in the fi eld of plasmid manufacturing (Aldevron, Anemocyte, Cobrabio, Delphi Genetics, Eurofi n, Eurogentech, Plasmid Factory, VGXI) usually offer both high quality product and GMP, addressing regulatory requirements applied at different clinical research level.



AnemocyteAnemocyte is a Biotech Manufacturing Organisation (BMO); a biotech company active in the fi eld of cell and gene Therapies (CGTs) that addresses CGT needs pro-actively offering one stop shop solutions and fostering exciting innovations.

Our business:• Process development and GMP capabilities (somatic cells, non-viral modifi ed cells, vesicles)

• Plasmids for viral vector manufacturing

• Project Exellula to address up to commercial scale needs for CGTs including viral vectors modifi ed cells

• Big data analysis of CGTs

Our experience:• More than 15 years of GMP manufacturing of CGTs and biological drugs

• More than 60 years of contract manufacturing within our group (Nine Trees Group)

Find out more at www.anemocyte.com

Phacilitate:ExchangePhacilitate:Exchange is the largest online advanced therapies community with contributors and members from all corners of the industry’s ecosystem. Phacilitate’s content hub provides access to premium, original market intelligence year-round, from videos and webinars to infographics and interviews.

We don’t report the daily news. We collaborate with leading minds to create resources that will help to the advanced therapies industry to drive commercialisation.

Find out where the industry is going next at www.phacilitate.co.uk

Anemocyte is a Biotech Manufacturing Organisation (BMO); a biotech company active in the fi eld of cell and gene Therapies (CGTs) that addresses CGT needs pro-actively offering one stop shop solutions and

• Process development and GMP capabilities (somatic cells, non-viral modifi ed cells, vesicles)

• Plasmids for viral vector manufacturing

• Project Exellula to address up to commercial scale needs for CGTs including viral vectors modifi ed

• Big data analysis of CGTs

Our experience:• More than 15 years of GMP manufacturing of CGTs and biological drugs

• More than 60 years of contract manufacturing within our group (Nine Trees Group)

Find out more at www.anemocyte.com

Phacilitate:ExchangePhacilitate:Exchange is the largest online advanced therapies community with contributors and members from all corners of the industry’s ecosystem. Phacilitate’s content hub provides access to premium, original market intelligence year-round, from videos and webinars to infographics and interviews.

We don’t report the daily news. We collaborate with leading minds to create resources that will help to the advanced therapies industry to drive commercialisation.

Find out where the industry is going next at www.phacilitate.co.uk

technology review: gene editing platforms - phacilitate.co.uk · cell types. simple process...

Documents