Charles Y. Cheung

pH-sensitive polymers that enhance intracellular drug delivery in vivo.

Cytosolic delivery from endosomes is critical for those drugs that are susceptible to attack by lysosomal enzymes, such as DNA, RNA, oligonucleotides, proteins and peptides. Therefore, we have designed pH-sensitive, membrane-disruptive polymers to enhance the release of drugs from the acidic endosomal compartment to the cytoplasm. We have found that one polymer in particular, poly(propylacrylic acid) (PPAA), is very effective at membrane disruption at pHs below 6.5, based on hemolysis studies. PPAA also significantly enhances in vitro transfections of lipoplex formulations in cell culture, and does so in the presence of as much as 50% serum. In this study, we have extended our in vitro hemolysis and cell culture studies to an in vivo murine excisional wound healing model. A pilot study with a green fluorescent protein (GFP)-encoding plasmid indicated that injection of formulations containing PPAA into healing wounds resulted in increased GFP expression. Subsequently, by administering sense and antisense DNA for the angiogenesis inhibitor thrombospondin-2 (TSP2), we were able to alter the wound healing response in TSP2-null and wild type mice, respectively. Our findings showed that when PPAA was added to lipoplex formulations, expression of TSP2 was enhanced in TSP2-null mice compared to control formulations. These results show that PPAA can enhance in vivo transfections and that inhibition of TSP2 expression may lead to improved wound healing. These results suggest that PPAA can provide significant improvements in the in vivo efficacy of drugs such as DNA.

Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles

The permeability barrier posed by cell membranes represents a challenge for the delivery of hydrophilic molecules into cells. We previously proposed that poly(2-alkylacrylic acid)s are endocytosed by cells into acidified vesicles and are there triggered by low pH to disrupt membranes and release the contents of endosomes/lysosomes to the cytosol. If this hypothesis is correct, these polymers could be valuable in drug-delivery applications. The present paper reports functional comparisons of a family of three poly(2-alkylacrylic acid)s. Poly(2-propylacrylic acid) (PPAA), poly(2-ethylacrylic acid) (PEAA) and poly(2-methylacrylic acid) (PMAA) were compared in red-blood-cell haemolysis assays and in a lipoplex (liposome-DNA complex) assay. We also directly examined the ability of these polymers to disrupt endosomes and lysosomes in cultured human cells. Our results show that: (i) unlike membrane-disruptive peptides, the endosomal-disruptive ability of poly(2-alkylacrylic acid)s cannot necessarily be predicted from their haemolytic activity at low pH, (ii) PPAA (but not PEAA or PMAA) potently facilitates gene transfection by cationic lipoplexes and (iii) endocytosed poly(2-alkylacrylic acid)s are triggered by luminal acidification to selectively disrupt endosomes (not lysosomes) and release their contents to the cytosol. These results will facilitate the rational design of future endosomal-disrupting polymers for drug delivery.

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Formulation of chitosan-DNA nanoparticles with poly(propyl acrylic acid) enhances gene expression

Poly(propyl acrylic acid) (PPAA) is a polymer specifically designed to disrupt lipid bilayer membranes within a sharply defined pH range. The pH sensitivity can be used to enhance the release of endocytosed drugs into the cytoplasmic compartment of the cell. By incorporating this polymer in a polymeric gene carrier, chitosan, the release of plasmid DNA from the endosomal compartment was enhanced. In vitro transfection studies confirmed that the incorporation of PPAA into the chitosan-DNA nanoparticles enhanced gene expression in both HEK293 and HeLa cells compared to chitosan nanoparticles alone. The dose and time at which PPAA was incorporated during the complex formation affected the release of DNA and transfection efficiency. The optimal dose of PPAA incorporated into the chitosan nanoparticles was determined to be 10 μg, corresponding to a PPAA/DNA weight ratio of 1: 1. At this dose, the ternary complexes are approx. 400 nm in size with a net negative surface charge of –17.4 mV. Intracellular trafficking studies confirmed the association of PPAA, DNA and chitosan at 24 h post-transfection and the subsequent release of DNA and PPAA from the chitosan at 48 h. The diffuse appearance of the majority of the DNA and the PPAA at later time points suggests that the PPAA triggered membrane disruption resulting in the release of DNA from the endosomal compartment. Finally, the lack of colocalization between PPAA and Lysotracker indicated that the PPAA-loaded nanoparticles were not trafficked through a lysosomal pathway. This study suggests the promising strategy of including PPAA in the formulation of polymer–DNA complexes for non-viral gene delivery.

Multifunctional pancreatic islet encapsulation barriers achieved via multilayer PEG hydrogels.

The diverse requirements for a successful islet encapsulation barrier suggest the benefit of a barrier system that presents differing functionalities to encapsulated cells and host cells. Initially, multifunctional hydrogels were synthesized via the sequential photopolymerization of PEG hydrogel layers, each with different isolated functionalities. The ability to achieve localized biological functionalities was confirmed by immunostaining of different entrapped antibodies within each hydrogel layer. Survival of murine islets macroencapsulated within the interior gel of two-layer hydrogel constructs was then assessed. Maintenance of encapsulated islet survival and function was observed within multilayer hydrogels over 28 days in culture. Additionally, the functionalization of the islet-containing interior PEG gel layer with cell–matrix moieties, with either 100 μg/ml laminin or 5 mM of the adhesive peptide IKVAV found in laminin, resulted in increased insulin secretion from encapsulated islets similar to that in gels without an exterior hydrogel layer. Finally, through cell seeding experiments, the ability of an unmodified, exterior PEG layer to prevent interactions, and thus attachment, between nonencapsulated fibroblasts and entrapped ECM components within the interior PEG layer was demonstrated. Together the presented results support the potential of multilayer hydrogels for use as multifunctional islet encapsulation barriers that provide a localized biologically active islet microenvironment, while presenting an inert, immunoprotective exterior surface to the host environment, to minimize graft–host interactions.

GIGI: an approach to effective imputation of dense genotypes on large pedigrees.

Recent emergence of the common-disease-rare-variant hypothesis has renewed interest in the use of large pedigrees for identifying rare causal variants. Genotyping with modern sequencing platforms is increasingly common in the search for such variants but remains expensive and often is limited to only a few subjects per pedigree. In population-based samples, genotype imputation is widely used so that additional genotyping is not needed. We now introduce an analogous approach that enables computationally efficient imputation in large pedigrees. Our approach samples inheritance vectors (IVs) from a Markov Chain Monte Carlo sampler by conditioning on genotypes from a sparse set of framework markers. Missing genotypes are probabilistically inferred from these IVs along with observed dense genotypes that are available on a subset of subjects. We implemented our approach in the Genotype Imputation Given Inheritance (GIGI) program and evaluated the approach on both simulated and real large pedigrees. With a real pedigree, we also compared imputed results obtained from this approach with those from the population-based imputation program BEAGLE. We demonstrated that our pedigree-based approach imputes many alleles with high accuracy. It is much more accurate for calling rare alleles than is population-based imputation and does not require an outside reference sample. We also evaluated the effect of varying other parameters, including the marker type and density of the framework panel, threshold for calling genotypes, and population allele frequencies. By leveraging information from existing genotypes already assayed on large pedigrees, our approach can facilitate cost-effective use of sequence data in the pursuit of rare causal variants.

rMAT-an R/Bioconductor package for analyzing ChIP-chip experiments

SUMMARY Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) has evolved as a popular technique to study DNA-protein binding or post-translational chromatin/histone modifications at the genomic level. However, the raw microarray intensities generate a massive amount of data, creating a need for efficient analysis algorithms and statistical methods to identify enriched regions. RESULTS We present a fast, free and powerful, open source R package, rMAT, that allows the identification of regions enriched for transcription factor binding sites in ChIP-chip experiments on Affymetrix tiling arrays. AVAILABILITY The R-package rMAT is available from the Bioconductor web site at http://bioconductor.org and runs on Linux, MAC OS and MS-Windows. rMAT is distributed under the terms of the Artistic Licence 2.0.

Poly(propylacrylic acid)-mediated serum stabilization of cationic lipoplexes.

The serum instability associated with cationic lipoplexes represents one of the major obstacles for the in vivo delivery of nonviral gene therapy vectors. Recently, we have shown that poly(propylacrylic acid) (PPAA), a pH-sensitive polyanionic polymer, can significantly improve the in vitro serum stability of DOTAP lipoplexes and enhance transfection (Cheung et al., Bioconjug. Chem. 12, 906 (2001)). We investigated this serum-stabilizing effect provided by PPAA using methods to identify the specific serum proteins that interact with DOTAP/DNA and DOTAP/DNA/PPAA lipoplexes and determined their modes of interaction with these lipoplexes. Studies showed that only low-density lipoprotein (LDL) caused significant decondensation of DNA from lipoplexes lacking PPAA, but that fully condensed DNA was retained within lipoplexes incorporating PPAA. Another major factor in the loss of transfection activity was due to the reduced cellular uptake of DOTAP lipoplexes upon exposure to serum, with bovine serum albumin (BSA) and high-density lipoprotein (HDL) acting as major contributors to this reduction in vector internalization. In contrast, lipoplexes containing PPAA maintained high levels of uptake into cells in the presence of these proteins. Transfection results generally concurred with the mechanistic studies, suggesting that maintaining effective cellular delivery of intact lipoplexes in the presence of serum proteins is important to retain high transfection efficiencies. These results indicate that the addition of PPAA as a ternary component in DOTAP lipoplexes can overcome some of the serum-related deficiencies encountered with these lipoplexes to provide efficient transfection.

Estimating and adjusting for ancestry admixture in statistical methods for relatedness inference, heritability estimation, and association testing.

It is well known that genetic association studies are not robust to population stratification. Two widely used approaches for the detection and correction of population structure are principal component analysis and model-based estimation of ancestry. These methods have been shown to give reliable inference on population structure in unrelated samples. We evaluated these two approaches in Mexican American pedigrees provided by the Genetic Analysis Workshop 18. We also estimated identity-by-descent sharing probabilities and kinship coefficients, with adjustment for ancestry admixture, to confirm documented pedigree relationships as well as to identify cryptic relatedness in the sample. We also estimated the heritability of the first simulated replicate of diastolic blood pressure (DBP). Finally, we performed an association analysis with simulated DBP, comparing the performance of an association method that corrects for population structure but does not account for relatedness to a method that adjusts for both population and pedigree structure. Analyses with simulated DBP were performed with knowledge of the underlying trait model.

Bioinspired polymers that control intracellular drug delivery

One of the important characteristics of biological systems is their ability to change important properties in response to small environmental signals. The molecular mechanisms that biological molecules utilize to sense and respond provide interesting models for the development of “smart” polymeric biomaterials with biomimetic properties. An important example of this is the protein coat of viruses, which contains peptide units that facilitate the trafficking of the virus into the cell via endocytosis, then out of the endosome into the cytoplasm, and from there into the nucleus. We have designed a family of synthetic polymers whose compositions have been designed to mimic specific peptides on viral coats that facilitate endosomal escape. Our biomimetic polymers are responsive to the lowered pH within endosomes, leading to disruption of the endosomal membrane and release of important biomolecular drugs such as DNA, RNA, peptides and proteins to the cytoplasm before they are trafficked to lysosomes and degraded by lysosomal enzymes. In this article, we review our work on the design, synthesis and action of such smart, pH-sensitive polymers.