Abstract:Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where… Show more
“…7, 70 In summary, efforts to improve complexation and subsequent gene delivery have resulted in the synthesis of CPs with amine groups intermittently interchanged with thiol moieties, 71 surface attachment of pendant hydroxyl groups, 51, 72 backbone alkylation, 73 and imidazole grafting (upon a chitosan backbone). 35 In addition to improved complexation, imidazole-functionalized CPs possessed pKa values of ~6, which is expected to facilitate enhanced gene delivery by polyplex escape from endosomal compartments (to be further explained below).…”
A key end goal of gene delivery research is to develop clinically-relevant vectors that can be used to combat elusive diseases such as AIDS. Despite promising engineering strategies, efficiency and ultimately gene modulation efficacy of nonviral vectors have been hindered by numerous in vitro and in vivo barriers that have resulted in sub-viral performance. In this perspective, we concentrate on the gene delivery barriers associated with the two most common classes of nonviral vectors, cationic-based lipids and polymers. We present the existing delivery barriers and summarize current vector-specific strategies to overcome said barriers.
“…7, 70 In summary, efforts to improve complexation and subsequent gene delivery have resulted in the synthesis of CPs with amine groups intermittently interchanged with thiol moieties, 71 surface attachment of pendant hydroxyl groups, 51, 72 backbone alkylation, 73 and imidazole grafting (upon a chitosan backbone). 35 In addition to improved complexation, imidazole-functionalized CPs possessed pKa values of ~6, which is expected to facilitate enhanced gene delivery by polyplex escape from endosomal compartments (to be further explained below).…”
A key end goal of gene delivery research is to develop clinically-relevant vectors that can be used to combat elusive diseases such as AIDS. Despite promising engineering strategies, efficiency and ultimately gene modulation efficacy of nonviral vectors have been hindered by numerous in vitro and in vivo barriers that have resulted in sub-viral performance. In this perspective, we concentrate on the gene delivery barriers associated with the two most common classes of nonviral vectors, cationic-based lipids and polymers. We present the existing delivery barriers and summarize current vector-specific strategies to overcome said barriers.
“…We next assessed the zeta potential of the NPs, an important index for average surface charge of the NPs in solution. [13a, 21] The NPs have a positive surface potential of 24.17±0.74 mV (Fig. 1D), compared to the negative potential of naked pDNA (-25.90±0.32 mV).…”
Given the number of monogenic ocular diseases and the number of non-monogenic degenerative ocular diseases for which gene therapy has been considered as a treatment, the development of effective therapeutic delivery strategies for DNA is a critical research goal. Here we generate, characterize, and evaluate non-viral nanoparticles composed of glycol chitosan (GCS) and plasmid DNA (pDNA). We show that these particles are stable, do not aggregate in saline, are resistant to DNases, and have a hydrodynamic diameter of ∼250 nm. We further show that the plasmid in these NPs maintains its proper conformation and can be released and expressed inside the cell. To determine whether these NPs would be suitable for intraocular use, pDNA carrying the ubiquitously expressed CBA-eGFP expression cassette was compacted and subretinally injected into adult WT albino mice. At post-injection (PI) day 14, we observe substantial GFP expression exclusively in the retinal pigment epithelium (RPE) in eyes treated with GCS NPs but not in uncompacted pDNA or vehicle (saline) treated eyes. We observe no signs of gross retinal toxicity and at PI-30 days, there is no difference in electroretinogram function between GCS NP-, pDNA-, or vehicle-treated eyes. These results suggest that with further development GCS NPs may be a useful addition to our available repertoire of genetic therapies for the treatment of RPE-associated diseases.
“…This causes osmotic swelling and eventual bursting of the endosome, releasing the nanocarrier into the cytosol [75]. Although numerous publications support this buffering technique for inducing endosomal escape [22, 76-80], its validity has been questioned. Several studies demonstrate that amine containing polymers do not induce changes in pH as previously suggested, and argue that the osmotic pressure built during endosome acidification is theoretically insufficient to cause membrane bursting on its own [81].…”
Section: Targeting the Intracellular Space: Delivering Biopharmaceutimentioning
New biopharmaceutical molecules, potentially able to provide more personalized and effective treatments, are being identified through the advent of advanced synthetic biology strategies, sophisticated chemical synthesis approaches, and new analytical methods to assess biological potency. However, translation of many of these structures has been significantly limited due to the need for more efficient strategies to deliver macromolecular therapeutics to desirable intracellular sites of action. Engineered nanocarriers that encapsulate peptides, proteins, or nucleic acids are generally internalized into target cells via one of several endocytic pathways. These nanostructures, entrapped within endosomes, must navigate the intracellular milieu to orchestrate delivery to the intended destination, typically the cytoplasm or nucleus. For therapeutics active in the cytoplasm, endosomal escape continues to represent a limiting step to effective treatment, since a majority of nanocarriers trapped within endosomes are ultimately marked for enzymatic degradation in lysosomes. Therapeutics active in the nucleus have the added challenges of reaching and penetrating the nuclear envelope, and nuclear delivery remains a preeminent challenge preventing clinical translation of gene therapy applications. Herein, we review cutting-edge peptide- and polymer-based design strategies with the potential to enable significant improvements in biopharmaceutical efficacy through improved intracellular targeting. These strategies often mimic the activities of pathogens, which have developed innate and highly effective mechanisms to penetrate plasma membranes and enter the nucleus of host cells. Understanding these mechanisms has enabled advances in synthetic peptide and polymer design that may ultimately improve intracellular trafficking and bioavailability, leading to increased access to new classes of biotherapeutics.
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