Gene Therapy


After more than 30 years of development, gene therapy has gradually become an important means to treat genetic diseases, infectious diseases, tumors and other diseases, which provides more options for diseases that cannot be solved by traditional therapies. At the same time, the rapid development of gene editing and the accumulated experience in the application of delivery vectors have also greatly promoted the progress of gene therapy. At present, more than 20 gene therapy products on the market in the world. As a new treatment method, gene therapy has a good prospect, but it still faces many challenges to continue to develop into mainstream therapy.

Mechanisms and basic strategies

  • Use normal genes to compensate for mutations in genes.

eg. In the treatment of hemophilia, normal coagulation factor VIII(FVIII) or coagulation factor IX(FIX) genes can be used to compensate for the mutated FVIII or FIX genes.

  • Repair mutated genes in the body.

eg. For single-base mutated genetic diseases, such as Tyrosinemia TYPE I, Sickle Cell Disease (SCD), Duchenne Muscular Dystrophy (DMD) can restore gene function by repairing mutant bases.

  • Inactivation/activation of abnormal pathogenic genes.

eg. Familial hypercholesterolemia, hereditary deafness can be treated by knocking out the pathogenic genes.

  • Introduction of new genes or modified genes into the body.

eg. The use of chimeric antigen receptor T-cell immunotherapy (CAR-T) for cancer. The ultimate goal of gene therapy is to make the normal gene expressed consistently in the patient's body. The transduction of target genes can be roughly divided into two types1.

Ex Vivo Delivery of Gene Therapy.

Fig.1 Ex Vivo Delivery of Gene Therapy. (High, KA.; Roncarolo, MG 2019)


For ex vivo transduction, the patient's somatic cells are isolated for in vitro culture, the target gene is transferred into the cell, the cell gene is modified in vitro, and then the modified cells are re-transfused into the patient to achieve the purpose of treatment. This approach requires a gene-delivery vehicle (or vector), the DNA that makes up the gene itself, and a technically sophisticated facility for processing the cells.


In vivo gene delivery resembles the delivery of other types of pharmaceutical agents. The vector–gene construct is stored frozen; it is then thawed and prepared by a pharmacist and is typically administered in an outpatient procedure2.

In Vivo Delivery of Gene Therapy

Fig.2 In Vivo Delivery of Gene Therapy. (High, KA.; Roncarolo, MG 2019)

Vectors for gene therapy

Selection of appropriate delivery tools is essential for the safe and effective implementation of gene therapy. At present, the main vectors used are divided into viral vectors3 and nonviral vectors4.

  • Viral vectors

About 70% of gene therapy regiments use viruses as delivery vectors. However, most viruses are pathogenic and must be engineered to retain only the functional components of their own DNA integration, while eliminating the original pathogenic functional components.

  • Retrovirus

Retrovirus is the first type of viral vector to be developed. Retrovirus only has a single infection after modification, so as to avoid its spread in human cells, but also greatly reduce the pathogenicity of virus itself.

  • Adenovirus (ADV)

ADVs can infect a variety of dividing and non-dividing human cells, and are applicable to almost all cell lines and primary cells. However, ADVs cannot express their target genes for a long time in highly dividing cells and require multiple infections to achieve their effect.

AAV has no pathogenicity to the host and has long-term and stable expression in a variety of tissues and cells. Recombinant adeno-associated virus (rAAV) vectors for gene therapy can stably express functional proteins isolated from host cell genes after introducing foreign genes into the host. Due to its advantages of high biosafety level, wide range of hosts, long expression time and low immunogenicity, it has attracted more and more attention and applications in gene therapy.

  • Lentivirus

Compared with ADV and AAV, lentiviruses can carry larger and more complex genomes and have the ability to integrate transgenes into the host genome for stable long-term expression of products. But at the same time, the feature of semi-random integration will also lead to the potential safety hazard of mutation.

  • Nonviral vector

Different from the viral vector, the particle size of the nonviral vector at the nanometer level contributes to the targeting and effectiveness of the vector.

  • Cationic polymer vector

Polyethyleneimine (PEI) is an early cationic polymeric vector, which can inhibit lysosomes and increase the positive charge in the acidic environment of phagophore, providing greater protection for DNA and helping to release DNA from phagophore without damage.

  • Liposome vector

Liposomes wrap foreign genes and bind them to the cell membrane. As the liposomes are similar to the cell membrane, the fusion occurs, making the foreign genes enter the cell. Liposome mediated cell transfection method has been relatively mature, this method is simple, no immunogenicity, low cytotoxicity, but the transfection efficiency is not high.

  • Nanoparticle vector

The exogenous gene transduction efficiency of nanomaterials is higher than that of liposomes, and its size is small, so it can be transported into various tissues with blood, which has great potential in gene therapy in vivo.

Types of gene therapy drugs, viral and non-viral delivery strategies, and disease treatment status of gene therapy drugs.

Fig.3 Types of gene therapy drugs, viral and non-viral delivery strategies, and disease treatment status of gene therapy drugs. (Xiuhua Pan.; et al., 2021)

Gene editing technology

At present, gene therapy programs mainly treat diseases by means of viral vector delivery of exogenous genes. Although certain progress has been made, some uncertain factors can induce immune response of the body to the vector, which makes the gene therapy programs always have other potential disease risks. The emergence of gene editing technology provides a new idea for humans to reduce these potential disease risks.

  • ZFNs

ZFNs consists of a zinc finger protein domain that determines its specificity and a DNA-cutting Fok I nuclease domain. This technique has already been used in mammalian cells, fruit flies, zebrafish, mice and plants. Since the construction of a specific zinc finger protein is time-consuming and laborious, and ZFNs has some off-target phenomena, how to effectively solve these two problems has become the main research direction of ZFNs technology in the future.

  • TALENs

TALE protein contains a series of highly conserved repeating units consisting of 33 to 35 amino acids. The 12th and 13th positions are two repeat-variable diresidue (RVD), which can recognize four different bases of DNA. TALE proteins bind DNA sequences specifically by RVD. Based on this discovery, Christian5 et al. fused TALEs with the dimerized Fok I to form TALENs.

  • CRISPR/Cas9

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to ZFN and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science6.

Schematic diagram of the CRISPR–Cas 9 system molecular mechanism

Fig.4 Schematic diagram of the CRISPR–Cas 9 system molecular mechanism. (Janik, E.; et al. 2020)

  • BE & PE

In 2016, David R. Liu and his team developed the base editing (BE) technology for the first time that can achieve single-base transformation without causing double strand breaking and without homologous template7. In 2019, Liu's team developed prime editor (PE) precision gene editing tool, which can realize arbitrary conversion, addition and deletion of 12 single bases and theoretically repair 89% of human genetic disease genes8.

Gene therapy drugs

Gene therapy has shown great potential in the treatment of major refractory diseases such as malignant tumors, infectious diseases, autoimmune diseases and rare diseases, and has gradually become a research hotspot. At the same time, it has greatly promoted the commercialization of gene therapy products. With the development of various gene therapy technologies, the safety and effectiveness of the carrier used for gene therapy products have been gradually improved, and a number of gene therapy products have been approved for marketing.

Table 1 Gene therapy drugs approved for marketing in the US and EU

Classification Name Indication Approved country Year
Gene interference Macugen Senile macular degeneration US 2004
Kynamro Homozygous familial hypercholesterolemia US 2013
Exondys 51 Duchenne muscular dystrophy US 2016
Spinraza Spinal muscular atrophy China & US 2019 & 2016
Defitelio Hepatic venular occlusive disease US & EU 2017
Gene insertion Glybera Lipoprotein lipase deficiency EU 2014
Imlygic Melanomas lesions US 2015
Strimvelis ADA-SCID EU 2016
Kymriah Acute lymphocytic leukemia US 2017
Yescarta DLBCL & PMBCL US 2017
Luxturna Vision loss due to RPE65 gene purity and mutation US 2017
Zolgensma Spinal muscular atrophy US 2019
Zynteglo Beta thalassemia EU 2019


  1. Dunbar, CE.; et al. Gene therapy comes of age. Science. 2018 Jan 12;359(6372):eaan4672.
  2. High, KA.; Roncarolo, MG. Gene Therapy. N Engl J Med. 2019 Aug 1;381(5):455-464.
  3. Lundstrom, K.; Viral Vectors in Gene Therapy. Diseases. 2018 May 21;6(2):42.
  4. Foldvari, M.; et al. Non-viral gene therapy: Gains and challenges of non-invasive administration methods. J Control Release. 2016 Oct 28;240:165-190.
  5. Christian, M.; et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010 Oct;186(2):757-61.
  6. Janik, E.; et al. Various Aspects of a Gene Editing System-CRISPR-Cas9. Int J Mol Sci. 2020 Dec 16;21(24):9604.
  7. Komor, AC.; et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 19;533(7603):420-4.
  8. Anzalone, AV.; et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Dec;576(7785):149-157.

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