44 Structure and Silencing Activity of Imidazole Containing Peptide siRNA Polyplexes Are Dependent on Hydrogen Bonding Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Soci[.]
CHEMICAL AND MOLECULAR CONJUGATES with down-regulated pro-apoptotic genes), which is sophistically controlled under healthy conditions Therefore, expressing a proapoptotic gene while simultaneously silencing the expression of the corresponding antagonistic gene can render synergistic cancer gene therapy This simultaneous, dual modal gene therapy can also significantly lower the levels of required genetic manipulation of each gene, greatly improving treatment safety In this study, viral/nonviral chimeric nanoparticles (ChNPs) were prepared with adeno-associated virus (AAV) in the core (to express a pro-apoptotic gene) with aciddegradable polyketal (PK) shell (to encapsulate siRNA against a prosurvival gene) AAVs are not known for any human diseases and can express a transgene either temporarily or permanently in the specific locations in human chromosome (low risk of random integration) Incorporating both cDNA (viral core) and siRNA (PK shell) in a nanoparticle ensures co-delivery to the same cells for synergistic effects The ChNPs are also designed for targeted intracellular localization of AAV and siRNA to the nucleus and the cytoplasm, respectively In addition, the PK shell shields the AAV core from immune responses during circulation, facilitates endosomal escape via acid-hydrolysis, and provides platform for facile conjugation of various modalities (e.g., targeting ligands and imaging contrast agents) The core-shell structure of the resulting ChNPs was confirmed by DLS and TEM Differentially ordered release of siRNA and the AAV core under intracellular stimuli-mimicking conditions was demonstrated by gel electrophoresis and nucleic acid quantification Targeted intracellular trafficking and localization of the AAV core and siRNA to the nucleus and the cytoplasm, respectively, were observed by confocal microscopy In vitro study with human B cells showed significantly enhanced transduction by ChNPs especially at low AAV concentrations, compared with unmodified AAV, along with simultaneously silenced expression of additional gene ChNP’s transduction ability was found intact in the presence of anti-AAV antibodies in the entire tested concentration range, proving efficient shielding of the AAV core by the PK shell Conjugation of sialic acid to the ChNPs surface enabled them to specifically bind and transduce CD22+ B cells Synergistically promoted apoptosis of Ph+ (BCR-ABL) human leukemia cells was also observed when they were incubated with ChNPs with the BIM (pro-apoptotic protein)expressing AAV core and Mcl-1 (pro-survival protein) siRNAencapsulating PK shell, while unmodified AAV and free siRNA at the corresponding doses resulted in negligible effects Preliminary in vivo studies also indicated that simultaneously tackling pro-apoptotic and pro-survival genes using novel ChNPs is a promising strategy to safe and efficient cancer gene therapy 43 Examining Cell-Penetrating Homochiral Cyclic Peptides as siRNA Non-Viral Transfection Agents Christiaan D King,1 Ria L Swanekamp,2 Bradley L Nilsson,2 David A Dean.1 Department of Pediatrics, University of Rochester, Rochester, NY; Department of Chemistry, University of Rochester, Rochester, NY Gene silencing via the siRNA degradation pathway has promising potential as a therapeutic strategy for a number of genetic and nongenetic disease states However as with gene therapy there are a number of barriers to successful delivery of siRNA, primarily the plasma membrane Unlike plasmid delivery, which requires nuclear import for transgene expression, siRNA needs only to be delivered to the cytoplasm to function properly in gene silencing There are a number of “carriers” one can use to transport DNA or siRNA into cells These include viral vectors and non-viral vectors such as lipoplexes, polyplexes, and cell penetrating peptides (CPPs) Although the use of peptides as carriers for either DNA or siRNA is not novel, the cellular dynamics of CPPs are enigmatic Recent data with HIV-Tat peptide suggests cyclisation of the peptide can enhance S18 membrane translocation potential Here we evaluate the amphipathic homochiral (D and L isoforms) cyclic peptides C[FKFE]2CG and C[WR]4CG and their potential use as non-viral siRNA transfection agents In Vivo and In Vitro When assessed for the ability to promote cellular uptake of fluorescently labeled molecules (plasmids, proteins, siRNA), the peptides facilitated uptake of siRNA and small drugs but not larger molecules, with the D-isoforms of both peptides outperforming their L-isoform counterparts However, when used to deliver functional siRNAs targeting the transcription factor TTF1 in A549 lung epithelial cells, the L-isoforms of both peptides mediated greater knockdown than the D-isoforms, although all carriers showed some degree of knockdown (30% to 70% relative to scrambled siRNA or no carrier controls) We next delivered TTF-1 siRNAs to mouse lungs by instillation of the siRNA (or scrambled siRNA) complexed by preincubation for 30 with the peptide and evaluated knockdown 48 hours later The relative activities of the peptide isoforms were similar to those seen in cultured cells, resulting in a maximal knockdown of 70% of the protein in total lung lysates Taken together, these results suggest that these peptides may be a useful means for siRNA delivery in living animals 44 Structure and Silencing Activity of Imidazole-Containing Peptide siRNA Polyplexes Are Dependent on Hydrogen Bonding Szu-Ting Chou,1,2 Kellie Hom,1 Lucas Tricoli,3 Jason Hustedt,3 Amy Lee,4 Joonil Seog,4 Jason Kahn,3 Qixin Leng,1 Daoning Zhang,3 Michael Shapiro,5 Archibald J Mixson.1 Pathology, University of Maryland Baltimore, Baltimore, MD; Chemical and Biomolecular Engineering, University of Maryland, College Park, MD; 3Biochemistry, University of Maryland, College Park, MD; 4Bioengineering, University of Maryland, College Park, MD; 5Pfizer Pharmaceuticals, Groton, CT Branched peptides containing lysines and histidines (HK) have been determined to be effective carriers for DNA or siRNA We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the polyplex In siRNA silencing experiments with mammalian cells, a 4-branched H3K(+H)4b peptide siRNA polyplex maintained silencing activity even with prolonged pre-incubation with serum (fig A) In contrast, siRNA in complex with 4-branched N3K4b, in which histidines were substituted with asparagines, showed a marked decreased in silencing activity with pre-exposure to serum Consequently, we explored the hypothesis that histidine might form non-covalent bonds with nucleic acids to enhance the stability of siRNA polyplexes To accomplish this, we initially compared the biophysical properties of H3K(+H)4b with N3K4b and polylysine Consistent with siRNA silencing experiments, gel electrophoresis analysis demonstrated that the HK siRNA polyplex maintained its integrity for more than 24 h incubation in 50% serum, whereas siRNA in complex to N3K4b or polylysine was degraded in a timedependent manner We next studied the thermodynamic profiles of various peptides binding to siRNA at pH 7.3 with isothermal titration calorimetry While polylysine and linear lysine-alanine peptide (A2K) interacted with siRNA resulting in an endothermic reaction, branched and linear lysine-histidine peptides (H3K(+H)4b and H2K) both exhibited an exothermic reaction This indicates that an important part of the exothermic interaction was based on non-ionic bond formation of histidines with siRNA To investigate the type of non-ionic bond, we studied the protonation state of the imidazole ring of a selectively N15 labeled H3K(+H)4b upon siRNA binding with heteronuclear single quantum coherence NMR The peak of N1protonated tautomers of imidazole (green boxes) shifted downfield (in the direction of deprotonation, represented by the green arrows in the fig B) by 0.5 to 1.0 ppm with addition of siRNA, providing direct evidence that uncharged histidines formed hydrogen bonds Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy INFECTIOUS DISEASES AND VACCINES with siRNA at physiological pH Moreover, unshifted N1 tautomers (black boxes) described the directionality of hydrogen bonds Taking together, these results indicate that histidine-rich peptides form nonionic bonds, and in particular hydrogen bonds, with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo This delivery approach also induced CSP- (0.6%) and LSA1- (1.5%) specific IFN production by hepatic CD8+ lymphocytes The immune responses conferred in mice translated to the NHP model Seroconversion was detected against all antigens (GMT OD=1): CSP (586), LSA1 (520), TRAP (2279), and CelTOS (1821) Potent cellular responses were detected by IFN ELISpot (2035 SFU) and in the CD4+ (0.6%) and CD8+ (0.7%) T-cell compartments Notably, the majority of antigen-specific CD8+ T cells were Granzyme B+ (0.7%) and T-bet+ (0.5%), suggesting the potential for cytotoxic and effector functions In conclusion, DNA+EP delivery elicits strong immune responses against multiple malaria antigens and merits further study in clinical trials 46 T-Cells Edited To Express the C34 Peptide from gp41 Heptad Repeat-2 Fused to CCR5 or CXCR4 Exhibit Robust Protection from Diverse HIV-1 Isolates Jianbin Wang,1 George J Leslie,2 Jennifer Duong,1 Kevin L Hua,1 Jenny J Yan,1 Beth Haggarty,2 Andrea P Jordon,2 Josephine Romano,2 Lei Zhang,1 Edward J Rebar,1 Philip D Gregory,1 James A Hoxie,2 Michael C Holmes.1 Sangamo Biosciences Inc., Richmond, CA; 2University of Pennsylvania, Philadelphia Infectious Diseases and Vaccines 45 Inducing Humoral and Cellular Responses to Multiple Sporozoite and Liver-Stage Malaria Antigens Using pDNA Bernadette Ferraro,1 Kendra T Talbot,1 Amritha Balakrishnan,1 Matthew P Morrow,2 Natalie A Hutnick,1 Devin J Myles,1 Devon J Shedlock,1 Nyamekye Obeng-Adjei,1 Jian Yan,2 Rachel Shiver,1 Amir S Khan,2 Maria Yang,2 Ami S Brown,2 Ulrike Wille-Reece,3 Ashley Birkett,3 Niranjan Y Sardesai,2 David B Weiner.1 University of Pennsylvania School of Medicine, Philadelphia, PA; Inovio Pharmaceuticals, Inc., Blue Bell, PA; 3The PATH Malaria Vaccine Initiative, Washington, DC A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against P falciparum (Pf) malaria Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four Pf antigens: CSP, LSA1, TRAP, and CelTOS Synthetic DNA sequences were designed with modifications to improve expression, and were delivered using electroporation (EP) Immunogenicity was evaluated in mice and non-human primates (NHP) and assessed by ELISA, IFN ELISpot, and flow cytometry DNA+EP delivery conferred robust antigen-specific IFN production in mice as measured by ELISpot (3172 SFU) and seroconversion against all antigens (GMT OD=1 >1,000) Antigen-specific cells were found in the CD4+ (1.5%) and CD8+ (2.9%) T-cell compartments Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy Recent advances in cell-based approaches to restore immune function and viral control in HIV-1 infected patients include the use of genetically modified autologous CD4 T-cells that can then be expanded ex vivo and adoptively reinfused However, there remains a need to develop robust strategies that protect cells from diverse HIV-1 isolates i.e R5-, X4-, and R5X4-tropic viruses In a novel approach to render autologous CD4 T-cells resistant to HIV-1, we evaluated the ability of a 34 amino acid peptide from the C-terminal heptad repeat-2 domain of gp41 (C34) to inhibit HIV-1 entry when fused to the amino terminus (NT) of either CCR5 (R5) or CXCR4 (X4) Firstly, C34-R5 or C34-X4 fusion constructs were stably expressed on the surface of CXCR4-/- SupT1 cells and shown to mediate chemotaxis to MIP1 or SDF1, respectively, demonstrating the physiologic functions of these receptors were maintained In contrast, HIV challenge experiments revealed that neither C34-coreceptor fusion supported HIV infection in stably or transiently expressing cells This effect was highly specific, as C34 peptides containing point mutations predicted to disrupt gp41 interaction were fully permissive for HIV entry Moreover, the C34 peptide conjugated to the NT of CD4 failed to inhibit HIV-1 entry, suggesting that positioning of the peptide was critical for blocking infection Remarkably, C34-R5 and C34–X4 were shown to potently inhibit HIV-1 infection/entry in a trans-dominant manner irrespective of Env tropism (i.e C34-R5 and C34-X4 could inhibit entry of both R5 and X4 tropic isolates) This broad and potent trans-dominant, heterologous inhibition of HIV-1 by C34-modified chemokine coreceptors represents a new and potentially powerful approach to engineer HIV-resistant autologous CD4 T-cells 47 Integrase-Defective Lentiviral VectorInduced Dendritic Cells: Clinical Development and Validation of CD4+/CD8+ T Cell Responses for Combination with Adoptive CMV-Specific T-Cell Therapy Anusara Daenthanasanmak,1 Constanca Figueiredo,1 Eliana Ruggiero,2 Manfred Schmidt,2 Arnold Ganser,1 Renata Stripecke.1 Hannover Medical School (MHH), Hannover, Germany; Nationales Zentrum fuer Tumorerkrankungen (NCT), Heidelberg, Germany Cytomegalovirus (CMV) reactivation or infection after hematopoietic stem cell transplantation (HSCT) is a major cause of mortality and morbidity Adoptive transfer of CMV-specific T S19 ... that histidine-rich peptides form nonionic bonds, and in particular hydrogen bonds, with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo This... (0.7%) and T-bet+ (0.5%), suggesting the potential for cytotoxic and effector functions In conclusion, DNA+EP delivery elicits strong immune responses against multiple malaria antigens and merits... Moreover, the C34 peptide conjugated to the NT of CD4 failed to inhibit HIV-1 entry, suggesting that positioning of the peptide was critical for blocking infection Remarkably, C34-R5 and C34–X4 were