Overview and vectors for gene therapy

Biopharma PEG https://www.biochempeg.com
Overview and Vectors For Gene Therapy
On January 25, 2022, Nature published an article listing seven technologies worthy of
attention this year. Targeted genetic therapies was on the list. The remaining six
technologies are: Fully finished genomes, Protein structure solutions, Quantum simulation,
Precise genome manipulation, Spatial multi-omics), CRISPR-based diagnostics.
An overview of gene therapy
Gene therapy refers to the delivery of exogenous genes with normal functions to target
cells in the human body with a certain carrier, and the purpose of treating diseases is
achieved by correcting the defective genes. If chemotherapy is a "symptom cure", then
gene therapy is a "cure for the root cause". With the gradual emergence of gene therapy
in clinical applications, it has become one of the most rapidly developing directions in the
field of biomedicine in recent years.
Most gene therapy is to obtain modified functional cells locally or in vitro, and then
transplant them into the patient. However, this method is limited in drug delivery and
cannot precisely deliver the drug to the target tissue. Of course, except for the liver,
because the liver has the function of filtering blood, in addition to intravenous infusion,
even subcutaneous injection can achieve specific targeted delivery to the liver. For gene
drug delivery in extrahepatic tissues, major pharmaceutical companies are making steady
progress to seize key technologies. Drug carrier is the key technology of gene therapy,
and commonly used carriers can be divided into viral vectors and non-viral vectors.
Gene Therapy Vectors
Depending on the source and nature of the vector, gene therapy vectors can be divided
into two categories: viral vectors and non-viral vectors. Viral vectors mainly include

lentivirus, adenovirus, retrovirus, adeno-associated virus, etc., and non-viral vectors
mainly include naked DNA, lipid carriers, polymer nanoparticles, and exosomes. Among
them, viral vectors are the main delivery vectors currently used, and about 70% of the
genetic drugs in clinical trials are viral vectors.
1. Viral Vectors
Viral vectors are the most commonly used vectors for gene therapy, mainly
because viral vectors can naturally infect cells. The genome of the virus includes a
coding region and a non-coding region. The genes in the coding region produce the
structural and non-structural proteins of the virus, while the non-coding region contains
cis-acting elements necessary for the replication and packaging of the virus. Gene
recombination technology can be used to modify the virus, eliminating the oncogene in the
genome and at the same time replicating the defective virus. Under normal circumstances,
in order to insert enough exogenous DNA into the virus, the unnecessary and essential
genes can also be deleted at the same time if necessary, so as to increase the capacity of
the viral vector for exogenous DNA.
Figure 1 Viral vectors for gene therapy

An ideal viral vector should have the following characteristics: it can encapsulate
exogenous genes and form virus particles; it can mediate the transfer and expression of
exogenous genes; it will not proliferate and spread in the environment, and will not cause
harm to the body. There are three main types of commonly used viral vectors:
Lentivirus (LV) vector, Adenovirus (ADV) vector and Adeno-associated virus (AAV)
vector. Adeno-associated virus is currently the most used vector.
(1) Lentiviral vectors
Lentivirus (LV) vectors are developed on the basis of HIV-1 (human immunodeficiency
virus type I). Lentiviral infection has the characteristics of integration, which can integrate
exogenous functional genes into the host chromosome, and the exogenous functional
genes become infectious virus particles under the action of virus encapsulation, so as to
achieve good gene therapy effect through stable and long-lasting expression.
After the lentiviral vector enters the cell, the carried genome is reverse transcribed into
DNA in the cytoplasm, and the reverse transcribed DNA enters the nucleus and integrates
into the cell genome. The DNA integrated into the cell genome can either generate small
RNA or be transcribed into mRNA for the expression of the target protein in the cytoplasm.
Lentivirus-mediated gene therapy can divide along with the division of the cell genome,
providing stable and efficient gene delivery.
(2) Adenovirus vectors
Adenovirus (ADV) vectors are the earliest human vector for gene delivery. Adenoviruses
are non-enveloped, double-stranded DNA, first isolated in the 1950s, and discovered in
the 1980s for gene delivery carrier potential. At present, no less than 60 types of
adenoviruses have been found, among which Ad5 (Adenovirus serotypes 5) type is
widely used as a gene delivery vector.

The delivery mechanism of adenovirus vectors is mainly receptor-mediated. The
recombinant adenovirus vectors enter the cell under receptor-mediated endocytosis, and
the genome carried by the adenovirus vectors enter the nucleus, but does not integrate
into the host cell genome, remaining outside the chromosome. Adenoviral vectors are
the most commonly used vaccine vectors and are less used in other areas.
(3) Adeno-associated virus vectors
Adeno-associated virus (AAV) vectors are non-integrated viral vectors (or the proportion
of gene integration ability is extremely low), which exist in an independent free form after
entering human cells and will not integrate into the host cell genome, thus reducing
related risks and showing good safety. Adeno-associated virus (ADV) is a class of
single-stranded DNA deficient viruses with the simplest structure. It has no envelope and
is shaped as naked 20-hedron particles. The scientific consensus is that it does not cause
any human disease and can infect different target organs according to the different serum.
Recombinant adeno-associated virus (rAAV) particles as gene therapy vectors have
successfully transduced mammalian cells since the early 1980s. Recombinant
adeno-associated virus particles bind to glycosylated receptors on the surface of host
cells, and enter cells to form endosome through clathrin-mediated endocytosis. The
subunit of viral capsid changes conformational changes after acidification, and the virus
carried by it disintegrates from the endosome and enters the nucleus. At this point, the
single-stranded DNA released from the capsid cannot be transcribed, requiring the
formation of double-stranded DNA with the assistance of DNA polymerase of the host
cell.

Figure 2 Approaches to gene therapy
Viral vectors have become a delivery tool for many gene therapies, and it has been
shown in many animal experiments that organ targeted gene delivery can be achieved
through high-throughput screening of suitable viral vectors and specific combination with
tissues. However, there are two difficult problems in viral vectors: one is that it is difficult
to mass produce viral vectors to meet the market demand; the other is that high drug dose
may stimulate immune response, leading to rapid degradation or neutralization of the
vector, and the safety needs to be further confirmed.
2. Non-viral vectors
The construction process of viral vectors is complicated and expensive, and after the viral
vectors enter the human body, immune responses will inevitably occur as the dose
increases, and some viruses even have off-target effects and carcinogenic risks. A series

of unresolved problems limit the development of viral vectors. At the same time, non-viral
vector technology has developed rapidly in recent years as a vehicle for novel gene
delivery therapies. Compared with viral vectors, non-viral vectors have their unique
advantages: the use of natural or semi-synthetic compounds, lower toxicity and
immunogenicity, and biodegradable properties reduce the risk of gene therapy; non-viral
vectors can be engineered and engineered, improve the targeted delivery efficiency of the
vector; the non-viral vector is easy to produce and process transformation, and the cost is
controllable.
(1) Lipid nanoparticles
In the past more than a year, lipid nanoparticles have become one of the hottest drug
delivery vectors due to the approval of mRNA COVID-19 vaccines. Lipid nanoparticles
have previously been used as non-viral vectors for gene delivery by actively fusing with
lipid cell membranes for delivery into cells. Relevant studies have shown that lipid
nanoparticles can be used as a substitute for viral vectors and have great potential for
tissue-specific targeted delivery, which can be widely used in the delivery of RNA
vaccines, RNAi, antisense nucleic acids and other drug molecules.
At present, lipid nanoparticles are used for gene therapy. The lipid nanomaterials
encapsulate the genome and directly enter the target cells for in vivo treatment. They can
be efficiently delivered without relying on viral vectors, and at the same time reduce the
risk of viral vector insertion into carcinogenesis. With the development of nanotechnology,
it has been possible to systematically screen lipid nanoparticles, by changing their
composition, physicochemical properties and biological properties, to change the
distribution of the genome they carry in the organism. At the same time, lipid nanoparticles
are also combined with genetic engineering techniques to maximize the therapeutic effect.
(2) Polymer nanoparticles

Polymer nanoparticles, as gene delivery vehicles, are usually combined with gene
editing technology. For example, Sarepta Therapeutics' polymer nanoparticle delivery
platform (NanoGalaxy) combined with Sarepta's gene editing technology to develop a
novel gene editing therapy for the treatment of neuromuscular diseases. Preliminary in
vivo results show that polymer nanoparticles deliver genomes to specific muscle tissues
after systemic administration without the assistance of targeted delivery of viral vectors.
The polymer nanoparticle delivery platform contains thousands of polymers with different
chemical properties, which can be selected according to the different targets to be
reached, and carry different load genomes. The polymer nanoparticles of the NanoGalaxy
technology platform can deliver DNA, RNA or CRISPR gene editing systems.
Figure 3 Working principle of NanoGalaxy delivery technology platform
(3) Exosomes
Exosomes are discoid vesicles with a diameter of 40-160 nm wrapped in lipid bilayers.
They are derived from multivesicular bodies formed by the invagination of intracellular
lysosomal particles. The outer membrane of multivesicles is fused with the cell membrane.
After being released into the extracellular matrix, exosomes are natural carriers of
intercellular communication, which have been developed as drug delivery vehicles at
present.

Compared with other delivery systems, exosomes have the following
advantages: as multifunctional carriers, exosomes can encapsulate and deliver various
biological macromolecules such as small RNA, mRNA, DNA, and proteins; exosomes
have the ability to cross physiological barriers , and can even cross the blood-brain barrier;
exosomes can be genetically engineered to modify their surface proteins to achieve
targeted delivery to specific tissues, avoiding toxic side effects caused by accumulation in
non-essential organs.
At present, exosome-based targeted delivery gene therapy has shown the advantages of
enhanced efficacy and improved safety, and other companies have combined lipid
nanoparticles and exosomes to develop a new generation of non-viral gene therapy.
In the history of gene therapy delivery, various delivery methods have been developed.
From viral vectors to non-viral vectors, from adenovirus to exosomes, with the
advancement and development of science and technology, gene therapy also benefits. It
is believed that with the continuous innovation of technology, gene therapy will bring more
possibilities for drug development.
Biopharma PEG is a worldwide leader of PEG linker supplier. We have been focusing on
the development of a full range of medical applications and technologies for nanocarrier
systems, including various types of nanoparticles, liposomes, micelles, etc. We are
committed to providing a variety of PEG-liposome derivatives, including mPEG, DSPE
lipids with different molecular weight and functional PEG. We can provide the
following PEG products that used in COVID-19 mRNA vaccines.
1
mPEG-N,N-Ditetradecylacetamide
(ALC-0159)
CAS No.
1849616-42-7
2 mPEG-DMG CAS NO.

160743-62-4
3 mPEG-CH2CH2CH2-NH2 ---
4 mPEG-OH
CAS NO.:
9004-74-4
5 mPEG-CM (mPEG-AA) ---
6 mPEG-DSPE
CAS NO.:
147867-65-0
7 mPEG-DPPE
CAS NO.:
205494-72-0
References:
[1] Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells
for Research and Gene Therapy.
[2] Adenovirus: the first effective in vivo gene delivery vector.
[3] AAV-Mediated Gene Therapy for Research and Therapeutic Purposes.

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