Croda Pharma

Lipid technology
for delivery ofgene-editing therapies
StephenPh.D.,
Croda
PharmaStephenBurgess,
Burgess,Ph.D.,
Croda
PharmaG

ene-editing technology is moving from the research lab
to the clinic — one of the most eagerly anticipatedbiopharma developments in recent history. One ofthe biggest hurdles to the development of successful gene-editing
therapeutics is effective delivery of the large and sensitive
components needed to execute the editing functions. Lipid-based
nanoparticles present promising solutions to these challenges.

The potential of therapeutic CRISPR
gene-editing
Gene-editing technologies are used tomanipulate DNA and RNA sequences by
cutting, inserting, or replacing genetic
sequences in the genome. Three primary
technologies have been developed to date:zinc finger nuclease (ZNF), transcription
activator-like effector nuclease (TALEN),
and clustered regularly interspaced shortpalindromic repeat (CRISPR).1 Initially, thistechnology was primarily used for research
purposes to identify genes involved in disease pathways — either assessing the consequences of a gene knockout/knockdown
or reversing a phenotype by introducing thegene for a functional protein. Today, however, many therapeutic candidates based on
gene-editing technologies are now advancing through the clinic.

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The developers of the first version ofThe Cas9 protein and sgRNA can be de-CRISPR/Cas9 gene-editing technology re-livered separately or complexed together asceived the 2020 Nobel Prize in Chemistrya ribonucleoprotein (RNP), which is the ac-

for their work. A host of advances have beentive species that performs the genome ed-

made since the original CRISPR editing

iting within the cell.1 Alternatively, plasmid

tools were introduced — primarily via genet-

DNA can be delivered to the cell, where it isic engineering — with some of the current

transcribed into messenger RNA and thencandidates leveraging second-generationtranslated into Cas9 protein. 2 Another op-technology and others relying on third-gen-tion involves the use of Cas9 mRNA, which

eration approaches that have addressedmust be translated into Cas9 protein oncesome of the earlier issues with the precision

it is inside the cell. DNA is more stable thanand persistence required for therapeuticmRNA, but the kinetics of plasmid delivery

applications. Further improvements are an-are slower than those for mRNA.

ticipated as candidates move through pre-Regardless of the approach, delivery isclinical and clinical development and morechallenging, owing to the large size of theknowledge is gained.

Cas9 protein and the overall CRISPR/Cas9

LNPs are attractive
because they have low
immunogenicity andprovide greater flexibility
with respect to thecargos for which they
can facilitate delivery
and the organs or cells
that can be targeted.

A number of companies that have beensystem, as the components must reach theexploring this approach are currently see-cell nucleus. Stability is also an issue. The

ing rapid acceleration of their programs.

low intracellular delivery efficiency mustdelivering Cas9 RNP into hard-to-transfectFor instance, Verve Therapeutics and In-

be overcome to achieve effective gene-edit-

cells, including induced pluripotent stem

tellia Therapeutics have begun clinical tri-

ing therapeutics.1 There are also concerns

cells. 2 It is inefficient for in vivo delivery,als. Many new companies are entering theabout the ability to safely and effectively de-however, so most uses have been in ex vivo

space as well, quite a number of them fo-liver repeated treatments, which for many

applications. Microinjection involves injec-

cused on in vivo therapies. As gene-editing

large tissues and organs will be necessary

tion of the CRISPR components into cells

systems are optimized and become moreusing current delivery technologies. 2 The

under a microscope and thus has limited

precise and efficient, the ability to addressneed for repeat administration has the po-throughput. In addition, while there is no

new and different diseases will become

tential to result in immunogenicity on thecargo size limitation, it does require spe-possible, and there will be continued move-

one hand and the development of immunity

cialized equipment and expertise and isment toward in vivo therapeutics.

on the other.

largely focused on the zygote stage, which

Two CRISPR-Cas9 components to deliver

Why LNPs are attractive delivery vehicles

Sonoporation combines ultrasound with in-Unlike more traditional gene therapies

Several different approaches have been em-

jection and has been investigated in a few

that involve delivery of a single gene of in-ployed for the delivery of CRISPR gene-ed-development products.

terest, CRISPR/CRISPR-associated systemiting tools, and they can generally be clas-

Biological delivery largely involves the(Cas9) gene-editing requires co-delivery ofsified as physical, biological, or chemical. 2

use of viral vectors and virus-like particles. 2

the Cas9 endonuclease protein and the sin-

Physical delivery methods include elec-With viral vector delivery, the CRISPR/

gle guide RNA (sgRNA) that targets the ed-

troporation, microinjection, and sonopo-Cas9-encoding sequences are integrated

itor protein to a specific genomic location.

ration. Electroporation can be effective at

into the viral genome, resulting in releaseis not appropriate for human therapeutics.

of the RNP complex into the targeted cells.1
There are safety concerns (e.g., mutations,carcinogenesis,immuneresponse)

andpackaging limitations with certain viralvectors. Exosomes (membrane-bound vesicles) are another natural alternative for
CRISPR/Cas9 delivery that are being explored because of their biocompatibility
and low immunogenicity.
Delivery of CRISPR gene-editing tools
can also be achieved with chemical methods, including liposomes, lipid nanoparticles(LNPs),

polymericnanoparticles,

gold nanoparticles, and other nanoscale
solutions.1,2 LNPs are attractive because
they have low immunogenicity and provide
greater flexibility with respect to the cargos for which they can facilitate delivery
and the organs or cells that can be targeted.1,3 They can also be formulated with

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LNPs increase
the efficiency ofin vivo CRISPR/Cas9
component delivery
and reduce toxicity
compared withviral vector technology
while maintaining
the necessary
precision to achieve
therapeutic effects.

lipid coating comprising lipid components

mulation and regulatory expertise, allowthat are biocompatible and make it easierthe company to meet its customers’ mostto cross the lipid membranes of cells. They

demanding formulation needs. The compa-

are highly efficient at sequestering and pro-ny is committed to enabling the next gener-

tecting the genetic material and enablingation of drug delivery systems.

its intercellular uptake. Even the non-nat-

Avanti Polar Lipids, now part of Crodaural ionizable lipids, depending on howPharma, has been supplying lipids for ther-

they are manufactured, are biocompatible

apeutic applications, including lipids used

and metabolizable and thus do not present

for gene therapies, for more than 30 years.

toxicity issues. In addition, the polymeric

Researchers investigating both viral vector

core means that less lipid material is need-and nonviral delivery systems have been re-ed, which further reduces the toxicity. Fur-liably provided with high-quality, well-char-thermore, it is possible to tether a targeting

acterized materials. Croda has added theagent — a peptide, protein, or antibody — toability to supply lipids at commercial scale

the lipid to enable the targeting of theseto Avanti’s development arm supporting

therapies to specific areas. The key issue,clinical programs.

then, is designing the polymeric core soIn addition to supplying materials current-

that it can sequester the large gene-editing

ly in use for the delivery of CRISPR/Cas9components while maintaining the desired

gene-editing components, Croda is pursuingdelivery efficiencies. Advances in the devel-— in cooperation with its customers — efforts

opment of polymeric cores for hybrid LNPs

to develop innovative lipid products that canbiodegradable components as lipid-likewill enable the efficient, targeted delivery

address some of the issues that have arisen

nanoparticles (LLNs) with reduced lipo-of greater quantities of genetic material

with LNPs for mRNA vaccines around PEG

some biological toxicity.

with reduced toxicity, making polymericlipids and other components. Working col-core design a key area of research and de-laboratively with clients on lipids for thesevelopment in LNP production.

and next-generation gene-editing therapeu-

In one recent example, a CRISPR-Cas9/
sgRNA system developed as a gene-editing

tics is how Avanti and Croda apply Smart sci-therapy to treat Duchenne muscular dystrophy (DMD) was achieved using a chemically

A long-term lipid innovations partner

defined LNP solution that enabled safe andCroda Pharma is a leading partner for therepeated injection of the therapy into skele-development of excipients and the supply

tal muscle tissue in a mouse model.4

ence to improve livesTM.

of high-purity materials for pharmaceuti-REFERENCES

Overall, LNPs increase the efficiency of incal formulations. The company is focused

1. Duan, Li et al. “Nanoparticle Delivery of CRISPR/Cas9 forvivo CRISPR/Cas9 component delivery andon empowering biologics drug delivery

Genome Editing.” Front. Genet. 12 May 2021.

reduce toxicity compared with viral vector

through its Adjuvant Systems and Small

2. Taha, Eman A., Joseph Lee, and Akitsu Hotta. “Delivery oftechnology while maintaining the necessary

Molecule, Protein, and Nucleic Acid Deliv-precision to achieve therapeutic effects.

ery platforms. With a wide range of solutions for both the human and animal health

Packaging, efficiency, and precision
challenges

markets, the pharmaceutical portfolio is

While LNPs are attractive as delivery vehi-

ma’s products, along with its in-house for-unsurpassed in its excellence. Croda Phar-

CRISPR-Cas tools for in vivo genome editing therapy: Trends andchallenges.” Journal of Controlled Release. 342: 345–361 (2022).
3. Ross, Colin J. D. et al. “Lipid-Nanoparticle-Based Deliveryof CRISPR/Cas9 Genome-Editing Components.” Mol.
Pharmaceutics. 19: 1669–1686 2022.
4. Kenjo, Eriya et al. “Low immunogenicity of LNP allows
repeated administrations of CRISPR-Cas9 mRNA into skeletal
muscle in mice.” Nature Communications. 12: 7101 (2021).

cles for CRISPR/Cas9 gene-editing components, there are challenges that must beovercome. One difficulty is determining howto package enough material into the LNPs.
Research is therefore focused on developing
LNP delivery vehicles that can carry larger
payloads. Increasing LNP delivery efficiency and precision are other primary goals.
With gene-editing, it is necessary to deliver
the components to a large number of cells;
high delivery efficiency is therefore necessary to enable cost-effective doses. Preventing off-target editing is equally crucial.

LNPs with lipid-coated polymer cores
Hybrid LNPs present a potential solution.
They have solid polymeric cores that sequester genetic material and an external

Smart science to improve lives™

ABOUT THE AUTHOR
Stephen Burgess, Ph.D.

Head of Lipid Technology and Nucleic Acid Delivery, Croda Pharma
Stephen Burgess, Ph.D., serves as Managing Director of Avanti PolarLipids, and he is Head of Lipid Technology and Nucleic Acid Delivery for
Croda Pharma, supporting the group’s vision to become the global leader
in empowering biologics delivery. Over the past 30 years, he has worked
with numerous researchers to develop novel lipids and lipid formulations toaddress specific experimental challenges and improve drug delivery. Hehas played a key role in a diverse list of projects, including drug delivery
development, gene therapy, wound healing, and vaccine development. His
background in lipid synthesis, lipid biophysical properties, and membrane
dynamics has been instrumental in resolving problems associated withthese projects.

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