Manual Vector Targeting for Therapeutic Gene Delivery

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Vector technology that was pioneered at CHOP led to the development of the first FDA-approved gene therapies, including Kymriah for B-cell acute lymphoblastic leukemia and Luxturna for inherited retinal disease. For safe and effective application of these vectors, researchers must have a complete picture of where the virus delivers its genetic cargo in the body. Conventional methods to define gene transfer rely on fluorescent reporter genes that glow under a microscope, highlighting cells that take up and express the delivered genetic material.

However, these methods reveal only cells with stable, high levels of the cargo. The new technology described in this study allows researchers to better detect where the cargo is expressed, even if it is expressed at extremely low levels, or only for a very short time. Perelman Center for Cellular and Molecular Therapeutics. Due to methodological limitations, many sites of low-level gene transfer have been missed.

Combining AAV with gene editing machinery requires a more sensitive method for safe and effective applications. To address this crucial gap in knowledge, Davidson and her lab developed a new AAV screening method that uses sensitive editing-reporter transgenic mice that are marked even with a short burst of expression or very low expression. In side-by-side comparisons with conventional screening methods, the new method radically redefines the true extent of AAV-mediated gene transfer.

According to the authors, this novel screening method will help improve the safety of AAV-gene editing approaches because it better defines sites where the vector expresses the modified gene. Importantly, because high and stable expression levels are not required for effective editing, dose levels that would not be ideal for more stable expression might work very well for genome editing. Additionally, this method expands the utility of the AAV platform by revealing new, never-before-described sites of gene transfer.

It also offers an opportunity to better understand the basic biology of AAV vectors and what is required for them to effectively deliver their genetic payload. To achieve long-term gene expression, targeting vectors must navigate a series of extracellular and intracellular obstacles Figure 1 before reaching nuclei of the target cells, where the therapeutic gene can be expressed RNA targeting does generally not require delivery to the nucleus. An ideal vector should protect its nucleic acid cargo from degradation while in transit. The degree of target-cell specificity required varies widely and in some applications delivery to a specific set of cells is essential.

Therefore, vectors are derivatized with ligands or antibodies to ensure targeting to a very specific range of cells. Depending on the cell type, the overall electrical charge, and the nature of the vector, its uptake occurs via endocytosis through different mechanisms: vectors targeted via receptor-ligand interactions show rapid internalization in contrast to the non-specific endocytosis Figure 1. Although a large number of cells typically internalize vectors albeit small amounts , most of it ends up degraded except in only a very small fraction of cells the vector is capable of escaping the endosomes.

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At this stage the therapeutic gene may or may not separate from the targeting system. Once released from endosomes, the target gene should be able to move through the cytoplasm towards the nucleus. Unfortunately, inefficient entry into the nucleus is the major limiting step in the development of non-viral gene delivery systems, and studies aiming to characterize the mechanism of cytoplasmic transport should facilitate design of improved vectors Suh et al.

Finally, therapeutic genes need to enter the cell nucleus. In dividing cells it is facilitated by the nuclear envelope disassembly during cell division. However, alternative strategies must be developed for the nuclear targeting in non-dividing cells. While viruses have evolved to tackle this and all the other obstacles, synthetic vectors may not have all the necessary functions. Therefore, it is necessary to take the cellular barriers into consideration in the rational design of better non-viral vectors.

The basic building block of non-viral gene delivery is plasmid DNA pDNA , which contains the therapeutic gene and the simplest way of targeting is a direct administration of naked pDNA. Various routes have been explored, including intravascular delivery, inhalation, and intramuscular injection Herweijer and Wolff, However, the efficiency of unprotected pDNA delivery is low and various mechanical and physical methods like the gene gun, hydrodynamic injection, and electroporation are used to improve it Heller and Heller, ; Trollet et al.

Enhancement of gene expression can be also achieved by targeting ultrasonic wave to the tissue after injection of pDNA Akowuah et al.

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In addition to physical targeting methods, various chemical carriers lipids and polymers are being used. These carriers are engineered to overcome the targeting obstacles described above. Their properties include pDNA condensation to protect it from nucleases, cell specific targeting, and increasing DNA delivery to the nucleus. Some carriers are used to act as a tissue depot slowly releasing pDNA to achieve a continuous or controlled expression. The charge-charge interactions between cationic lipids and negatively-charged pDNA result in the formation of lipid-pDNA complexes lipoplexes.

These could form micelles or, under specific conditions, vesicles liposomes. However, lipid-based gene delivery has crucial limitations, including low stability of the lipoplexes significant manufacturing issue , toxicity to some cell types, and limited colloidal stability, especially upon systemic administration.

For these reasons there is an increased interest in using polymers for gene delivery. Because of the flexibility of polymer chemistries it should be possible to engineer multiple functionalities required for efficient gene delivery while maintaining biocompatibility, facile manufacturing, and robust and stable formulation.

Polymers in use include both off-the-shelf materials and specifically designed molecules.

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These can be grouped in two main categories: ionic and non-ionic. Application of polycations is also based on the charge interaction between negatively charged phosphate groups of nucleic acids with positively charged amines or amidines residing in the polymer leading to the compaction of DNA into spherical complexes called polyplexes.

In contrast to polycations, non-ionic polymers such as poly N -vinyl pyrrolidone and pluronic block copolymers do not rely on DNA condensation to enhance gene expression. Pluronics improve both levels and duration of expression when used with naked pDNA in muscles. However, recent data demonstrated that Pluronics act as biological response modifiers, i. Consequently, some safety aspects of their administration should be re-considered and the mode of action of non-ionic polymers in general requires further studies. Because of their relatively high gene-delivery efficiency and good biocompatibility, polyamidoamine dendrimers and, more recently, chitosan are also interesting candidates in gene-delivery studies.

Polyethylenimine PEI , however, has been the most extensively studied cationic polymer and has been shown to effectively target a variety of tissues. Endosomes are acidified by active transport of protons from the cytosol.

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Polymer containing protonable amines can act as a proton-sponge, causing transport of more protons to reach the desired endosomal pH. As the accumulation of protons is balanced by an influx of Cl - , the increased ion concentration ultimately causes osmotic swelling, rupture of the endosome, and release of polyplexes into the cytosol.

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However, this effect may be also responsible for high PEI cytotoxicity — endolysosomal bursts would release proteolytic enzymes. Therefore, there needs a less aggressive escape strategy that involves using endosomolytic peptides that have membrane-disrupting preferably reversible activities in weak acidic conditions. Forgot your username? Enter your email address below and we will send you your username. Human Gene Therapy Vol. Kah-Whye Peng Search for more papers by this author. Frances J. Morling Search for more papers by this author.

Progress and problems with the use of viral vectors for gene therapy

Francois-Loic Cosset Search for more papers by this author. Gillian Murphy Search for more papers by this author. Russell Address reprint requests to: Dr. Reducing off target viral delivery in ovarian cancer gene therapy using a protease-activated AAV2 vector platform. Stimulus-responsive viral vectors for controlled delivery of therapeutics.


Adeno-associated virus AAV vectors in cancer gene therapy. Intracellular delivery of nanocarriers and targeting to subcellular organelles. Tunable Protease-Activatable Virus Nanonodes. Gene therapy, science fiction or science fact? Development of Latent Cytokine Fusion Proteins.

Library screening and receptor-directed targeting of gammaretroviral vectors. Designer nanoparticles: incorporating size, shape and triggered release into nanoscale drug carriers. Viral vectors: from virology to transgene expression. Gene therapy methods in bone and joint disorders. Matrix metalloproteases: Underutilized targets for drug delivery.


Targeted retroviral vectors displaying a cleavage site-engineered hemagglutinin HA through HA—protease interactions. Adult Stem Cells and Gene Therapy. Gene Therapy to the Nervous System. Viral gene therapy strategies: from basic science to clinical application. Retroviral display of urokinase-binding domain fused to amphotropic envelope protein. Gelatinase-mediated migration and invasion of cancer cells. Targeting cytokines to inflammation sites. Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met.