Synthetic delivery vectors have the potential to address many of the limitations of viral vectors, particularly with respect to safety.
For systemic delivery of DNA, both lipid-based vectors and polymer-based vectors have been intensively investigated in experimental animals and in clinical trials.
The potential of mRNA for therapeutic protein expression in vivo has been investigated as an alternative to DNA-based gene therapy owing to its unique advantages. Recent advances in chemical modifications of mRNA reduce stimulation of the immune system and improve stability when it is delivered in vivo.
Small interfering RNA (siRNA) has great therapeutic potential, as it can silence nearly any targeted gene after introduction into cells. Lipid- and polymer-based siRNA nanoparticles and conjugate systems enable successful delivery of chemically modified siRNAs in humans.
Levels of microRNA (miRNA) can be restored through the introduction of synthetic miRNAs or mimics as miRNA replacement therapy. One miRNA mimic is currently undergoing evaluation in a Phase I clinical trial.
Delivery of genome editing systems — including zinc-finger proteins, transcription activator-like effectors and CRISPR–Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated) systems — facilitates gene editing at desired sites in the genome. Recent proof-of-concept studies in model organisms have shown that this approach may be used to cure genetic diseases, which is in contrast to the temporary expression or random insertion of a DNA fragment in conventional gene therapy.
Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.
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The authors thank R. L. Bogorad, E. A. Appel, O. F. Khan, O. Veiseh, Y. Dong and G. Sahay for discussion. H.Y. is supported by the US National Institutes of Health (NIH) Centers for Cancer Nanotechnology Excellence and the Harvard–MIT (Massachusetts Institute of Technology) Center of Cancer Nanotechnology Excellence (5-U54-CA151884-04). R.K. is supported by the US National Science Foundation Graduate Research Fellowship (grant 1122374). A.A.E. is supported by the US National Heart, Lung, and Blood Institute, NIH, as a Program of Excellence in Nanotechnology (PEN) Award (contract HHSN268201000045C). This work was supported partly by the Koch Institute Support (core) Grant P30-CA14051 from the US National Cancer Institute. The authors acknowledge the service to the MIT community of the late S. Collier.
D.G.A. is a cofounder of CRISPR Therapeutics. D.G.A. has a research grant with Alnylam Pharmaceuticals and a research grant with Shire. J.R.D. is a shareholder of Alnylam Pharmaceuticals.
Single-stranded RNA viruses that use reverse transcriptase to transcribe their RNA into DNA, which can be integrated into the host genome. The viral DNA can be transcribed, translated and packed into new viruses. Replication-defective retroviruses are commonly used for gene therapy purposes. Most of these retroviruses are only active in dividing cells.
A subclass of retroviruses that are active in non-dividing cells. The replication-defective viruses are used both as a research tool to introduce a gene in vitro or in vivo and for gene therapy. They infect cells with high efficiency and introduce stable expression.
DNA viruses that do not integrate into the host genome and that usually do not replicate during cell division. Many of them trigger fast immune responses. For gene therapy purposes, they are usually applied in conditions in which temporary expression of proteins is required.
- Adeno-associated viruses
(AAVs). DNA viruses that have very low but measurable genome integration rates and that are used as vectors for gene therapy. They are able to infect both dividing and non-dividing cells with high efficiency and long persistence. Although they have small packing capability, they are preferred for gene therapy owing to their low immunogenicity and low cytotoxicity.
- Zinc-finger proteins
(ZFPs). DNA-binding proteins that consist of tandem arrays of zinc-fingers, which are protein structure motifs that contain one or more zinc ions. Engineered zinc-fingers have been shown to recognize three specific base pairs of DNA sequences and can be assembled in tandem to recognize specific nucleic acid sequences. The process of engineering is difficult and requires expertise.
- Transcription activator-like effectors
(TALEs). Proteins that were first discovered in Xanthomonas spp. bacteria and that bind to promoter sequences in host plants to facilitate infections. They contain a repeat domain of 34 amino acids. Two critical amino acids in each repeat allow targeting of specific DNA bases. TALEs can be engineered in a time-consuming process by assembling repeat domains to recognize specific DNA sequences.
(Clustered regularly interspaced short palindromic repeat–CRISPR-associated). Defense systems against foreign DNA in bacteria and archaea, in which a short CRISPR RNA (crRNA) is used to guide the Cas nuclease to a specific target DNA sequence. These systems have been optimized to function in mammalian cells with high efficiency. The engineering process to target various DNA sequences is straightforward, and the cost is low.
Vesicles of various sizes with a lipid bilayer that can encapsulate small molecules or large molecules such as small interfering RNA or DNA. By fusing to the cell membrane, they facilitate delivery of the liposome contents in vitro and in vivo.
Vesicles produced using co-polymers, which are made of two or more monomers, to allow both hydrophilic and lipophilic ability. They can encapsulate small molecules, proteins and DNA to form particles of various sizes, and are used for drug delivery systems in vitro and in vivo.
- Cell-penetrating peptides
Small peptides that can translocate across plasma membranes. By covalent or non-covalent binding of these peptides to small molecules, proteins, DNA, small interfering RNA or even nanoparticles, they could facilitate intracellular delivery of various types of molecules.
- Scaffold/matrix attachment regions
(S/MARs). Anchor points of the genomic DNA for the chromatin scaffold. They are found at introns or borders of transcription units, where they have an important role in separating these units and regulating gene expression.
A sequence-specific recombinase that mediates recombination between 2 specific 34-bp sequences, which allows insertion of DNA into another DNA molecule or into the genome at specific sites.
A transposon system composed of a transposon and a transposase, which recognizes transposon-specific sequences on both ends of a vector and integrates the content into different DNA molecules or genome.
- Sleeping Beauty
A transposon system reconstructed from DNA copies of salmon. The transposase was engineered to facilitate robust and stable gene transfer.
A commercial reagent that delivers small interfering RNAs into various types of cells in vitro with high efficiency. As a cationic lipid formulation, it can also be used to deliver microRNA antagonists and mimics, as well as mRNAs.
- Glomerular filtration barrier
A blood filtration interface in the kidney that allows free passage of small ions such as sodium and potassium but that retains large proteins. The cutoff to pass this barrier is ~70 kDa, and naked small interfering RNAs (~13 kDa) can thus be filtered through the kidney.
A restriction endonuclease with a DNA-binding domain at the amino terminus and a nonspecific DNA cleavage domain at the carboxyl terminus. Dimerization of two FokI is required to cleave DNA. When it is fused to zinc-finger proteins (ZFPs) or to transcription activator-like effectors (TALEs), FokI will function when a pair of ZFPs or TALEs binds to DNA within short distance. This strategy allows increased specificity of sequence recognition.
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Yin, H., Kanasty, R., Eltoukhy, A. et al. Non-viral vectors for gene-based therapy. Nat Rev Genet 15, 541–555 (2014). https://doi.org/10.1038/nrg3763
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