Delivery of
Gene Therapy

Delivery of genes to target cells is critical to effective gene therapy. This module reviews viral and non-viral vectors that can deliver genetic material to target cells.

Gene Delivery Systems1

  • Developing a gene therapy is a complex, multi-step process – a key element of which is the selection of an appropriate delivery system, or vector1
  • For a given therapeutic goal, the gene delivery system (vector) should offer1,2:
  • Specificity for the intended target

  • Sustained transgene expression

  • Low immunogenicity

Specificity for the Intended Target

Ideally, gene therapy systems would specifically target the appropriate cell type for the disease being treated2

However, vector distribution is influenced by a number of parameters2

Off-target uptake by other cells will be variable, and affects the tolerable levels of toxicity associated with treatment2

Detrimental Overexpression

Overexpression and non-targeted expression of the MECP2 gene is detrimental in Rett syndrome3

Preferential Overexpression

However, high expression levels of factor IX are preferential in haemophilia B, where overexpression is not generally a concern4

Delivery Challenge

Developing delivery systems that are targeted to the cell type of interest is a challenge2

One potential strategy is the use of tissue-specific promotors to drive expression in target tissues4

Sustained Transgene Expression

Matching Duration

The duration of transgene expression should match the time needed to treat the disease2

The target-cell division rate may have an effect on the duration of expression, especially when using non‑integrating viral vectors, whose DNA can be lost in cell division2

Sustained expression can be halted by epigenetic modifications to the vector genome, such as the silencing of promoter sequences2,5

Continuous Expression

Genetic diseases, such as haemophilia B, usually require life-long transgene expression2,6

This can be achieved by engineering the gene therapy to promote stable transgene expression after administration of the vector, or by re‑administration of treatment2

Limited Expression

However, acquired disorders such as infections or cancer may require shorter periods of transgene expression2

 Low Immunogenicity

Administration of gene therapy leads to activation of the host immune response, which can be directed against the transgene product and/or vector particles2

This could lead to serious side effects, such as cytokine release syndrome7,8, and could limit the viability of the transduced cells2

Viral Vector and Non-Viral Delivery Systems

Non-Viral Delivery Systems1,2,9,10

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Non-viral delivery systems comprise two basic components2:

  1. the DNA/RNA molecule
  2. the delivery constituent

Pros

  • Low immunogenicity due to lack of viral components
  • No limit on the size of the DNA that can be delivered
  • Cost-effective

Cons

  • Have a lower delivery efficiency, mostly due to their large size and susceptibility to endosomal degradation2
  • Low, transient expression of transgene

Viral Vectors1,2,9,11

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Viral vectors

  1. Viruses are often used as vectors because they can deliver the new gene by entering the target cell
  2. The viruses are modified so they can't cause disease when used in people

Pros

  • High delivery efficiency
  • Long-term gene expression

Cons

  • Potential safety concerns (e.g. immunogenicity, oncogenicity)
  • Limited payload (kb) capacity
  • Challenges with the manufacturing process
  • Until efficiency and sustainability of delivery can be improved in non-viral vectors, viral vectors will remain the systems of choice for transgene delivery, despite their associated safety concerns12

  • Approximately 70% of gene therapy trials and all approved agents use viral vectors due to their delivery efficiency and minimised potential safety concerns10

Non-viral delivery systems include physical and chemical methods2

Overview of Physical Non-Viral Delivery Methods9

Physical methods of gene delivery are based on using physical force to allow transient penetration of the cell membrane, thereby facilitating delivery of the genetic material into the target cells9,13

Delivery Methods Key Mechanism  Advantage Limitation
Naked plasma/plasmid DNA (pDNA) – direct delivery by injection Endocytosis Safety, simplicity Low transfection efficiency
Gene gun High-pressure helium stream Flexibility, low cytotoxicity, good efficiency Shallow penetration
Electroporation Enhancement of cell membrane permeability Good efficiency, repeatable Tissue damage, accessibility of electrodes to internal organ limited
Ultrasound + microbubble Enhancement of cell membrane permeability Safety, flexibility Low efficiency
Magnetofection Pinocytosis and endocytosis Flexibility, low cytotoxicity Transient transfection

Overview of Chemical Non-Viral Delivery Methods9

Chemical vectors can be classified as inorganic particles, lipid-based, polymer-based, and peptide-based9

They may be designed to protect the genetic material, target particular cells, enhance delivery, disintegrate from the DNA/RNA in the cytosol, or deliver sustained or controlled release of the therapeutic gene9

Delivery Methods Key Mechanism  Advantage Limitation
Inorganic molecule Endocytosis Easy production, storage stability, surface functionalization Low efficiency
Lipoplexes (lipid-based) Endocytosis, DNA condensation Safety, low cytotoxicity Low-to-medium efficiency, some results in immunogenicity
Polyplexes (cationic polymer-based) Endocytosis, DNA condensation, protein sponge effect Safety, low cytotoxicity Complement activation, low efficiency, cytotoxicity

Viral Vectors Used in Gene Therapy

Viral vectors are naturally occurring viruses that have been modified14

Most of the original viral genes have been replaced with the desired transgene13

The removal of viral genes means that it does not replicate or trigger the same immune response as the wild-type virus13

*Lentivirus vectors are derived from the retrovirus class of viruses15

Retroviral Vector

Applications of retroviral vectors10,13

  • Severe combined immunodeficiency
  • Cancers
  • Retroviral infections, e.g. HIV
  • Neurologic disorders, e.g. Huntington’s disease, motor neurone disease, Parkinson’s disease, Alzheimer’s disease
  • Familial hyperlipidemia

Axicabtagene ciloleucel is an ex vivo gene therapy product that has received regulatory approval for the treatment of B-cell lymphoma. It comprises autologous T cells that are genetically modified by retroviral transduction to express an anti-CD19 chimeric antigen receptor16

Name
Genetic material11 RNA
Packaging capacity11,17 8 kb
Tissue tropism17 Dividing cells only
Inflammatory potential11 Low
Safety11 Oncogenic potential
Main limitations11,17 Only transduces dividing cells; integration might induce oncogenesis in some applications
Main advantages17 Persistent gene transfer in dividing cells

Lentiviral Vector

Applications of lentiviral vectors*

  • Neurologic disorders, including motor neurone disease, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, spinal injury13
  • Lysosomal storage diseases13
  • Used for ex vivo gene transfer in the central nervous system and for treatment of cancer8,13

Tisagenlecleucel is an ex vivo gene therapy product that has received regulatory approval for the treatment of acute lymphoblastic leukaemia and B-cell lymphoma. It comprises autologous T cells that are genetically modified using a lentiviral vector to encode an anti-CD19 chimeric antigen receptor8

Name
Genetic material11 RNA
Packaging capacity11,17 8 kb
Tissue tropism17 Broad
Inflammatory potential11 Low
Safety11 Oncogenic potential
Main limitations11,17 Integration might induce oncogenesis in some applications
Main advantages17 Persistent gene transfer in most tissues

Adeno-associated Viral Vector

Applications of AAV vectors11,13,17,18

  • Spinal muscular atrophy
  • Hemophilia A and B
  • Neurodegenerative disorders
  • Retinal diseases
  • Muscular dystrophies
  • Cancer
  • Cardiovascular diseases
  • Metabolic diseases
  • Hepatitis C

Due to the good safety profile and the broad range of target tissues, use of AAV vectors in gene therapy clinical trials has been increasing19

Voretigene neparvovec-rzyl is an in vivo gene therapy product that has received regulatory approval for the treatment of inherited retinal disease. It is a live, non-replicating AAV that has been genetically modified to express the human RPE65 gene20

Name
Genetic material11 Single-stranded DNA
Packaging capacity11,17 <5 kb
Tissue tropism17 Broad, with the possible exception of hematopoietic cells. Different AAV serotypes have different tissue tropisms
Inflammatory potential11 Low
Safety11 Non-pathogenic; generally low immunogenicity
Main limitations11,17 Small packaging capacity limits the range of therapeutic genes
Main advantages17 Low- or non-inflammatory; non-pathogenic

Delivering Viral Vectors Containing the Transgene to Target Tissues

Gene therapy clinical trials using viral vectors use two methods to deliver therapeutic DNA to target tissues:

  • In vivo (direct) delivery:

    Direct injection of transgene-containing viral vector into the patient, where it then transduces the target tissues

Involves direct injection of viral vector into the target tissues21

Avoids the practical issues of ex vivo cell-based gene therapies22

In November 2018, the in vivo gene therapy product, voretigene neparvovec, was approved in Europe for the treatment of an inherited retinal disease3

It is administered via subretinal injection by a surgeon experienced in intraocular surgery23

Figure adapted from Figure 1, Collins M, Thrasher A. Proc R Soc B2015;282(1821):20143003.

  • Ex vivo (cell-based) gene delivery

    Modification of patient’s cells in culture by viral vectors followed by cell expansion and injection into the patient

Involves the modification of cells in culture (ex vivo) by viral vectors, followed by cell expansion and
re-injection into the patient21

Practical and regulatory challenges are associated with cell collection, culture, manipulation, and transplantation22

In 2018, two ex vivo gene therapy products were approved in Europe for the treatment of acute lymphoblastic leukaemia and relapsed or refractory large B-cell lymphoma4,5

Figure adapted from Figure 1, Collins M, Thrasher A. Proc R Soc B 2015;282(1821):20143003.

  1. Carvalho M, et al. Front Med 2017;4:182.
  2. Kay MA. Nat Rev Genet 2011;12(5):316–328.
  3. Clarke AJ, Abdala Sheikh AP. Orphanet J Rare Dis 2018;13:44.
  4. Powell SK, Rivera-Soto R. Discov Med 2015;19(102):49–57.
  5. Gray SJ, et al. Hum Gene Ther 2011;22:1143–1153.
  6. Nathwani AC, et al. N Engl J Med 2014;371:1994–2004.
  7. YESCARTA [package insert]. Santa Monica, CA; Kite Pharma, Inc.; 2017.
  8. KYMRIAH [package insert]. East Hanover, NJ; Novartis Pharmaceuticals Corporation; 2018.
  9. Ramamoorth M, Narvekar A. J Clin Diagn Res 2015;9(1):GE01–6.
  10. Chira S, et al. Oncotarget 2015;6:30675–30703.
  11. Lukashev AN, Zamyatnin Jr AA. Biochem (Moscow) 2016;81(7):926–936.
  12. Wang D, Gao G. Discov Med 2014;18:67–77.
  13. Nayerossadat N, et al. Adv Biomed Res 2012;1:27.
  14. Nayak S, Herzog RW. Gene Ther 2010;17:295–304.
  15. Chira S, et al. Oncotarget 2015;6:30675–30703.
  16. YESCARTA [package insert]. Santa Monica, CA; Kite Pharma, Inc.; 2017.
  17. Lundstrom K. Diseases 2018;6(2):42.
  18. Chira S, et al. Oncotarget 2015;6:30675–703.
  19. Colella P, et al. Mol Ther Methods Clin Dev2017;8:87–104.
  20. LUXTURNA [package insert]. Philadelphia, PA; Spark Therapeutics, Inc.; 2017.
  21. Collins M, Thrasher A. Proc R Soc B2015;282(1821):20143003.
  22. Dunbar CE, et al. Science 2018;359:175.
  23. FDA News Release. December 19, 2017. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss. Available at: "https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm589467.htm. Accessed January 14, 2019.
  24. FDA News Release. December 19, 2017. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss. Available at: href="https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm589467.htm. Accessed January 14, 2019.
  25. FDA Press Release. August 30, 2017. FDA approves tisagenlecleucel for B-cell ALL and tocilizumab for cytokine release syndrome. Available at: href="https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm574154.htm. Accessed February 13, 2019.
  26. FDA Press Release. October 18, 2017. FDA approves axicabtagene ciloleucel for large B-cell lymphoma. Available at: https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm581296.htm. Accessed February 13, 2019.