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
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.
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 Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy.
 Adenovirus: the first effective in vivo gene delivery vector.
 AAV-Mediated Gene Therapy for Research and Therapeutic Purposes.