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The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

A series of pyridinium cationic lipids was designed, synthesized and characterized. These lipids varied in the lipophilic part, bearing C9 to C20 saturated, unsaturated, straight and branched hydrocarbon chains. The lipid shape parameter was calculated from the molecular structure of these lipids based on the partial molar volumes of the atoms, and standard bond lengths and bond angles, using fragment additive methods. The shape parameter controls the lamellar/hexagonal phase balance in lipoplexes of the lipid with deoxyribonucleic acid (DNA). The lipid phase behaviour of the lipoplexes was derived from small-angle X-ray scattering experiments and was successfully correlated with the calculated lipid shape parameter. The synthesized pyridinium lipids were co-formulated (1:1) with 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) as the co-cationic lipid in 1:1 ratio, and the mixed cationic lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. The effect of variation in cationic lipid structure and lipoplex formulation on the transfection of nucleic acid (β-galactosidase and green fluorescent protein (GFP)) into CHO-K1 cells and the cytotoxicity of these formulations was assessed. Initial studies on the synthesized lipids bearing saturated and terminally unsaturated C16 chains showed that a Transfection Index (TIPSV) which encompasses the variation in the lipid shape parameter, the phase packing in a hexagonal lipoplex and the partition of these lipids into the lipoplex successfully correlated with transfection efficiency. To further investigate the effect of the variation of the partition of these lipids to the lipoplex, transfection studies were performed on a series of pyridinium lipids with straight saturated and unsaturated chains of varied lengths, with similar shape parameters but varied partition coefficients (clogP). The correlation of these experimental transfection data with the initial TIPSV was unsuccessful, but the data suggested that chain length as it relates to chain mixing and chain melting behaviours of pure lipids played a role in transfection. A refined transfection index (TIPSVM) was proposed which contained terms for the lipid shape parameter, the phase packing into a hexagonal lipoplex, the partition of these lipids into the lipoplex and a chain melting term. TIPSVM gave an acceptable correlation with the experimental transfection efficiency for the range of compounds. Additional experimental transfection data were obtained for compounds with widely variable lipid shape parameters, either as pure compounds, blends of two pure compounds, or statistically produced mixtures of mixed-chain compounds. Although very short-chain compounds (C9) and very lipophilic compounds (C20) performed poorly, the results from the blends allow the assessment of the role of the shape parameter in the TI. Since the shape parameter and the volume filling term are both calculated with the same molecular parameter, the experimental work demonstrated that only one of these terms is required. Thus a three parameter transfection index (TIPVM) was proposed and found to correlate with the entire set of comparable data. A Quantitative structure–activity relationship (QSAR) study was done on the cytotoxicity of the transfection formulations utilized. The toxicity of the synthesized pyridinium lipids was shown to correlate with the shape parameter, the lipid mixture partition co-efficient (clogP) and the charge ratio of the lipoplex formulation. Taken together, the developed transfection index TIPVM and the cytotoxicity correlation uncovered can be used in the design of low-toxicity, high activity pyridinium lipids for transfection of DNA. / Graduate / pariapz@uvic.ca

Restriction endonucleases occur ubiquitously among bacteria, archaea and in viruses of certain unicellular algae, and they are usually accompanied by a modification enzyme of identical specificity; together, the two activities form a restriction-modification (R-M) system- the prokaryotic equivalent of an immune system. More than 3,800 R-M enzymes have been characterized so far and they manifest 262 unique recognition specificities. These enzymes represent the largest family of functionally related enzymes. Based on the number and organization of subunits, cofactor requirements, catalytic mechanism, and sequence specificity, restriction enzymes have been classified into different types, Types I, II, III, and IV. R-M systems are important model systems for studying highly specific DNA-Protein interactions and serve as excellent systems for investigating structure-function relationship and for understanding the evolution of functionally similar enzymes with highly dissimilar sequence. In bacteria, DNA methyltransferases (MTases) associated with R-M systems protects the host DNA from cleavage by the cognate restriction endonuclease recognizing the same sequence and provides the integrity of host cell genome against foreign DNA invasion. The modification MTases catalyses the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on the chemistry of the methylation reaction catalyzed, DNA MTases are classified as C5 enzymes (endocyclic MTases), which transfer the methyl group to C5 position of cytosine, and N6 and N4 enzymes (exocyclic amino MTases), which transfer the methyl group to the exocyclic amino group of adenine or cytosine, respectively. DNA MTases of all three types contain conserved regions, which are responsible for catalysis and AdoMet binding, and variable regions known as target recognition domains (TRD), which determine the substrate specificity of a particular enzyme. Ten conserved amino acid motifs (I–X) are found in C5 MTases. Exocyclic DNA MTases are subdivided further into six groups (namely α, β, γ, ζ, δ and ε), according to the linear arrangements of three conserved motifs, the AdoMet-binding domain (FXGXG), the TRD (target recognition domain) and the catalytic domain (D/N/S)PP(Y/F). Base flipping has been proposed as a general mechanism used by all MTases in which the target base to be methylated is rotated 180º out of the DNA into a catalytic domain (motif IV). EcoP15I restriction enzyme (R.EcoP15I) belongs to the Type III restriction-modification (R-M) family. These enzymes are composed of two subunits, Res (Restriction) and Mod (Modification). The Mod subunit alone functions as a DNA methyltransferase in presence of AdoMet and magnesium and determines the specificity for restriction and methylation, whereas restriction activity requires the cooperation of both the Res and Mod subunits. EcoP15I methyltransferase (M.EcoP15I), a homodimeric enzyme catalyzes the transfer of a methyl group from AdoMet to the second adenine residue in the recognition sequence, 5’-CAGCAG-3’, in presence of magnesium ions. M.EcoP15I belongs to the β-subfamily of N6-adenine methyltransferases. In addition to the two highly conserved sequence motifs, FXGXG (motif 1) involved in AdoMet binding and DPPY (motif IV) involved in catalysis, the amino acid residues of the region 355-377 contains a PD(X)n(D/E)XK-like motif involved in metal binding. A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-Specific Endonuclease An interesting aspect of M.EcoP15I is that the methylation requires magnesium and magnesium binding to the PD(X)n(D/E)XK-like motif participates in base flipping. The PD-(D/E)XK superfamily of Mg2+-dependent nucleases were initially identified in structurally characterized Type II REases and later found in many enzymes involved in DNA replication, recombination and repair. The charged residues from the catalytic triads are implicated in metal ion mediated DNA cleavage. In EcoP15I DNA methyltransferase, a PD(X)n(D/E)XK like motif is present in which the partially conserved proline is replaced by methionine (MD(X)18(D/E)XK). Using site-directed mutagenesis methionine at 357 was changed to proline (M357P), which resulted in the formation of a Mg2+ binding/catalytic motif similar to several Mg2+-dependent endonucleases. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. The mutant protein specifically binds to the recognition sequence 5’-CAGCAG-3’ and cleaves DNA in presence of Mg2+. The engineered EcoP15I-M357P is an active, sequence-dependent restriction endonuclease that cleaves DNA 10/1 nucleotide away from its recognition sequence in the presence of Mg2+. Unlike the holoenzyme, R.EcoP15I, the engineered endonuclease neither requires AdoMet or ATP nor requires two sites in the inverted orientation for DNA cleavage. It is of potential interest to use such an engineered enzyme as a genetic manipulation tool. Dimerisation of EcoP15I DNA Methyltransferase is Required for Sequence Recognition and Catalysis In the cell, after each round of replication, substrate for any DNA MTase is hemimethylated DNA and therefore, only a single methylation event restores the fully methylated state. This is in agreement with the fact that most of the DNA MTases studied exist as monomers in solution. The peculiar feature of M.EcoP15I is that it methylates only one strand of the DNA, at the N6-position of the adenine residue. Earlier studies using gel filtration and glutaraldehyde cross-linking demonstrated that M.EcoP15I exists as dimer in solution. However, the significance of dimerisation in the reaction mechanism of EcoP15I MTase is not clear. Therefore, experiments have been performed to determine whether M.EcoP15I could function as a monomer and the significance of dimerisation, if any, in catalysis. Towards this a homology model of the M.EcoP15I was generated by “FRankenstein monster” approach. Residues D223, V225, and V392, the side chains of which are present in the putative dimerisation interface in the model were targeted for site-directed mutagenesis. These residues were mutated to lysine and their importance was studied. Methylation and in vitro restriction assays showed that the triple mutant was catalytically inactive. Interestingly, the mutations resulted in weakening of the interaction between the monomers leading to both monomeric and dimeric species. M.EcoP15I was inactive in the monomeric form and therefore, dimerisation might be the initial step in its function. This must be required for positioning of the target base of the DNA in the active-site pocket of the M.EcoP15I. A part of this interface may be involved in site-specific DNA binding. Dimerisation of M.EcoP15I is, therefore, a prerequisite for the high-affinity substrate binding needed for efficient catalysis. Understanding the role(s) of Amino and Carboxyl-terminal Domains of EcoP15I DNA Methyltransferase in DNA Recognition and Catalysis N-terminal and C- terminal domains (NTD and CTD) of proteins are known to play many important roles such as folding, stability, dimerisation, regulation of gene expression, enzyme activity and substrate binding. From the modeled dimeric structure of M.EcoP15I, it was hypothesized that N- and C-termini are in close proximity with each other. In addition, it was predicted that each monomer can bind to AdoMet and DNA. Towards understanding the role(s) of the N- and C-terminal domains of M.EcoP15I in its structure and function, N-, and C-terminal deletions were created. Interestingly, deletion of N-terminal 53 amino acids and C-terminal 127 amino acids from of EcoP15I MTase converted the dimeric enzyme to a stable, monomeric protein that was structurally stable but enzymatically inactive. Each monomer could bind single-stranded DNA but dimerisation was required for double-stranded DNA binding and methylation. This indicated that amino acids at the N- and C-termini are important for maintaining a proper dimeric structure for M.EcoP15I functions. Therefore, it can be proposed that in a complex three-dimensional structure, the NTD and CTD should be properly maintained in order to execute its function, including dimerisation and DNA binding. However, since the 3D structure of M.EcoP15I has not yet been determined, the biochemical, biophysical and bioinformatics approaches may serve to provide useful information on the relative contributions of the electrostatic forces and hydrophobic contacts to the structural stability. Understanding the structural organization and folding of M.EcoP15I is crucial to elucidation of the mechanism of action.

L’utilisation de bactéries lactiques (BL), telle que Lactococcus lactis (LL), comme vecteur de transfert d’ADN, constitue une stratégie prometteuse dans la mesure où elles sont considérées sans risque pour la santé. Des souches sauvages (wt) ou recombinantes de LL ont été décrites comme capables de transférer un plasmide dans des cellules épithéliales in vitro et in vivo. Cependant, les mécanismes d'action grâce auxquels certaines souches de LL ont la capacité de transférer de l’ADN plasmidique sont toujours inconnus. C’est pourquoi, nous avons décidé de construire une nouvelle souche recombinante de LL exprimant l’internaline mutée (mlnlA,) à partir de la souche pathogène Listeria monocytogenes, de manière à comprendre par quel procédé l’ADN est transféré à des cellules eucaryotes. Nous avons détecté l’expression de mInIA par FACS et montré que la souche LLmInIA était plus invasive que la souche sauvage wt après co-incubation avec des cellules épithéliales intestinales (IECs) non confluentes ou polarisées. La microscopie confocale confirme ces propriétés d’invasivité de la souche LL-mLnLA capable de transférer plus efficacement le vecteur d’expression eucaryote codant pour l’allergène de la β-lactoglobuline, pValac :BLG, in vitro dans des IECs et dans des cellules dendritiques (DCs). La souche LL-mInIA a aussi la capacité de transférer le vecteur pValac:BLG à des DCs à travers une monocouche de IECs différenciées. Des essais in vivo montrent que des bactéries invasives du genre Lactococcus ont tendance à augmenter l’expression de BLG chez la souris. De plus, il est montré qu’une souche non invasive de LL, ou la souche invasive LL-mInIA, stimulent la sécrétion de la cytokine pro-inflammatoire IL-12 dans des DCs, et que, in vivo, après des essais d’immunisation oraux ou intra nasaux, la souche LL non invasive oriente la réponse immunitaire plutôt vers le type 1, alors que la souche LL invasive génère une réponse de type 2 chez des animaux immunisés. Tous ces résultats apportent un nouvel éclairage sur le mécanisme d’assimilation des lactocoques en tant que vecteurs de transfert de molécules actives. / The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.

The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.

Die Entwicklung einer mukosal verabreichbaren, effektiven DNA-Vakzine gegen Infektionskrankheiten oder Tumorerkrankungen auf der Basis invasiver attenuierter Bakterien ist eine vielversprechende Alternative zu bisherigen parenteralen Strategien der genetischen Vakzinierung. Innerhalb dieser Arbeit wurden Salmonellen-Impfstämme für die orale Übertragung eines eukaryontischen Expressionsplasmids mit dem kleinen Oberflächenantigen des Hepatitis-B-Virus (HBsAg) als Modellantigen optimiert. Die kontinuierliche Sezernierung von Plasmiden als filamentöse Phagenpartikel wurde als ein erster Ansatz getestet, um mit lebenden Bakterien eine DNA-Vakzine innerhalb infizierter Zellen freizusetzen. Die Salmonellen-vermittelte Phagensekretion in der Wirtszelle ist jedoch nicht effizient genug, die Expression des Transgens zu vermitteln. Alternativ wurde ein Ansatz gewählt, durch eine spontan induzierte Lyse der Impfbakterien, Plasmid-DNA in die Wirtszelle zu übertragen. Dazu wurde ein neuartiges bakterielles Autolysesystem etabliert, basierend auf einem Zwei-Phasen-Expressionssystem und von Bakteriophagen abgeleiteten Lysedeterminanten. Dieses System ermöglicht erstmals die kontinuierliche Freisetzung von Plasmid-DNA und Proteinen aus einzelnen, lysierenden Salmonellen innerhalb einer sonst gesunden bakteriellen Gesamtpopulation. Innerhalb infizierter COS7-Zellen führt die Freisetzung des porenformierenden Proteins Listeriolysin O durch autolytische Salmonellen zur Zerstörung der Vakuole, in der die Impfbakterien replizieren, und erleichtert somit den Transfer der Plasmid-DNA aus den Bakterien in das Zytoplasma der Wirtszelle. Die Lysedeterminante und die eukaryontische Expressionskassette für HBsAg wurden auf einem Plasmid kombiniert, sowie eine Kassette zur konstitutiven Expression des Histon-ähnlichen Proteins aus Thermotoga maritima (TmHU) in ein solches Konstrukt integriert. TmHU stabilisiert die Plasmiderhaltung unter nicht selektiven Bedingungen und besitzt das Potential, die Effizienz der DNA-Translokation innerhalb der Wirtszelle zu erhöhen. Durch die orale Gabe optimierter autolytischer Impfbakterien konnte eine potente HBsAg-spezifische Antikörperantwort sowie eine zytotoxische zelluläre Antwort induziert werden. Bereits die einmalige Gabe der autolytischen Bakterien induzierte eine höhere antigenspezifische Antikörperantwort, als die herkömmliche intramuskuläre DNA-Vakzine. Das im Rahmen dieser Arbeit entwickelte Konzept autolytischer Salmonellen stellt also eine neuartige, effiziente Strategie für den mukosalen DNA-Transfer dar. Die Übertragung des Konzeptes der Autolyse auf andere bakterielle Trägersysteme ist möglich und kann zur Erweiterung des Anwendungspektrums bakterieller Vektoren beitragen. / The development of an effective mucosal DNA vaccine against infectious diseases or tumors based on invasive attenuated bacteria is a very promising alternative to common parenteral routes of genetic vaccination. This work aimed at the optimization of Salmonella vaccine strains for the oral delivery of an eukaryotic expression plasmid encoding the small Hepatitis B Virus surface antigen (HBsAg), here used as model antigen. The continuous secretion of plasmids as filamentous phage particles was first tested as a mean for the delivery of the DNA vaccine by living bacteria inside infected host cells. However, Salmonella-mediated phage secretion inside cells did not suffice for the induction of transgene expression. As alternative approach, inducible spontanous lysis of bacteria was used to mediate the release of plasmid DNA into host cells. For this purpose a novel bacterial autolytic system was established on the basis of a two-phase expression system and lysis determinants derived from bacteriophages. This system allows for the first time the continuous release of plasmid DNA and proteins from only few lysing Salmonella within an otherwise healthy bacterial population. Inside COS7 cells the release of the pore-forming protein listeriolysin O by autolytic Salmonella mediates the destruction of the Salmonella-harbouring vacuole, thereby facilitating the transfer of plasmid DNA from bacteria into the host cell cytoplasm. The lysis determinant was combined with the eukaryotic expression cassette for HBsAg on one plasmid. In addition, a cassette for the constitutive expression of TmHU, a histon-like protein derived from Thermotoga maritima, was integrated in such vector. TmHU stabilizes the plasmid propagation in the absence of selective pressure and has the potential to increase the efficiency of plasmid translocation inside the host cell. The oral administration of the optimized autolytic bacteria stimulated a potent HBsAg-specific antibody response as well as a cytotoxic cellular response. Already a single inoculation of the oral vaccine induced a higher specific antibody response than the conventional intramuscular DNA vaccine. Therefore the concept of autolytic Salmonella carrier strains developed in this work constitutes a novel efficient strategy for mucosal DNA delivery. The transfer of this concept to other bacterial carriers is possible and may widen the application field for bacterial vectors.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition. Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play also a prominent role in plasmid DNA delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53 deficient HCT116p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.