Combinatorial Modification of Degradable Polymers Enables Transfection of Human Cells Comparable to Adenovirus

Abstract

Viral gene therapy has high efficacy, but is plagued by serious safety risks, production and manufacturing challenges, and other limitations including nucleic acid cargo capacity. [1] In contrast, non-viral gene delivery systems, while addressing these challenges, remain less effective.[2] Here we develop end-modified poly(b-amino ester)s, easy-to-synthesize degradable polymers, that are able to deliver DNA to primary human umbilical vein endothelial cells (HUVECs) at levels comparable to adenovirus at a Multiplicity of Infection (MOI) between 100 and 500, and two orders of magnitude better than the commonly used non-viral polymeric vector, polyethylenimine (PEI). Interestingly, small structural changes were found to have dramatic effects on multiple steps of gene delivery including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression. In vivo, these polymer modifications dramatically enhance DNA delivery to ovarian tumors. We believe the development of polymeric vectors with gene delivery efficacy comparable to adenovirus could set a new benchmark in nonviral transfection capability. Numerous polymeric materials have been used for gene delivery including poly(L-lysine), polyethylenimine, poly(amidoamine) dendrimers, poly(a-[4-aminobutyl]-L-glycolic acid), chitosan, cyclodextrin, and others. [2,3] While significant strides have been made in improving delivery, efficacy remains generally low, particularly for primary cells in the presence of serum.[4] Poly(b-amino ester) s are promising materials that bind and self-assemble with DNA to form stable nanoparticles that effectively enter cells, escape the endosomal compartments, and degrade via hydrolytic cleavage of backbone ester groups. [5][6][7][8] While good in vitro and in vivo activity has been described, [5,9] structural diversity of existing poly(b-amino ester) was limited by chemical requirements of conjugate addition. [5][6][7][8]10] We hypothesized that the exploration of an expanded chemical space through combinatorial modification of poly(b-amino ester) s could optimize performance. To this end, and to better understand structure-function relationships, we synthesized a library of end-modified poly(b-amino ester) s using three base diacrylate-terminated polymers and twelve amine end-capping reagents (Fig. 1). Chemical methods were developed to allow a simple, one step modification of base polymers with several different amine capping agents (see methods). In this way, the combined effects of internal structure and amine termination on poly(b-amino ester) transfections could be systematically assessed. Once synthesized, polymers were characterized by 1 H NMR and GPC (see methods).Three different polymers were chosen for end modification: C32, D60 and C20 (Fig. 1B). High-throughput screening studies have identified polymer C32 as the most effective poly(bamino ester) to date for gene delivery. [5,6] Another polymer, D60 is also an effective gene delivery agent, but with a structure significantly different from...

A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

“…This leads to smaller nanoparticles with increased cellular uptake. We find that end-modified PBAEs have gene delivery efficacy comparable to lentivirus 38 and adenovirus 35 (Figure 10) for transfection of HUVECs in vitro. In comparison to the previous "gold standard" for polymeric transfection, 25 kDa polyethylenimine, the PBAE nanoparticles presented here have 2 orders of magnitude higher efficacy while simultaneously having 2 orders of magnitude lower toxicity.…”

“…To accomplish this, we synthesized a library of end-modified PBAEs using three diacrylateterminated base polymers and 12 amine monomers as end-capping reagents. 35 A one-step reaction was used to conjugate the new terminal groups to the base polymers and the combined effects of internal structure and terminal structure on PBAE efficacy were systematically assessed ( Figure 9A, B). Lead polymer C32 was end-capped with an expanded library of 36 different amines by Zugates et al to show wider structure/function relationships ( Figure 9C).…”

“…We find that end-modified PBAEs have gene delivery efficacy comparable to lentivirus 38 comparison to the previous "gold standard" for polymeric transfection, 25 kDa polyethylenimine, the PBAE nanoparticles presented here have 2 orders of magnitude higher efficacy while simultaneously having 2 orders of magnitude lower toxicity. 21,35 We believe that these polymeric vectors highlight the strength of a combinatorial polymer library approach, and they could set a new benchmark in nonviral transfection capability. …”

Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles

“…Combined stem cells and gene therapy may further stimulate angiogenesis by producing desired angiogenic and antiapoptotic factors, but safe and efficient gene delivery to stem cells has been challenging (20,21). To overcome this hurdle, combinatorial polymer synthesis and high-throughput screening have been used to facilitate the development of nonviral gene delivery systems (22). We have developed biodegradable nanoparticulate polymeric vectors that can deliver DNA into human stem cells with high efficiency and minimal toxicity (15).…”

“…We have developed biodegradable nanoparticulate polymeric vectors that can deliver DNA into human stem cells with high efficiency and minimal toxicity (15). End modification of the polymers were found to have dramatic effects on multiple steps of gene delivery, including the DNA binding affinity, nanoparticle size, intracellular DNA uptake, and final protein expression (22,23). To our knowledge, gene transfection efficiency (≈35%) using these end-modified polymer nanoparticles (15) was the highest for MSCs in serum-containing transfection conditions compared with previously reported methods using electroporation (16%), poly(L-lysine)-palmitic acid (17%) (24), or commercially available transfection reagents such as FuGene (3%) and DOTAP (5%) (25).…”