mRNA vaccines expressing malaria transmission-blocking antigens Pfs25 and Pfs230D1 induce a functional immune response.

Malaria transmission-blocking vaccines (TBV) are designed to inhibit the sexual stage development of the parasite in the mosquito host and can play a significant role in achieving the goal of malaria elimination. Preclinical and clinical studies using protein-protein conjugates of leading TBV antigens Pfs25 and Pfs230 domain 1 (Pfs230D1) have demonstrated the feasibility of TBV. Nevertheless, other promising vaccine platforms for TBV remain underexplored. The recent success of mRNA vaccines revealed the potential of this technology for infectious diseases. We explored the mRNA platform for TBV development. mRNA constructs of Pfs25 and Pfs230D1 variously incorporating signal peptides (SP), GPI anchor, and Trans Membrane (TM) domain were assessed in vitro for antigen expression, and selected constructs were evaluated in mice. Only mRNA constructs with GPI anchor or TM domain that resulted in high cell surface expression of the antigens yielded strong immune responses in mice. These mRNA constructs generated higher transmission-reducing functional activity versus the corresponding alum-adjuvanted protein-protein conjugates used as comparators. Pfs25 mRNA with GPI anchor or TM maintained >99% transmission reducing activity through 126 days, the duration of the study, demonstrating the potential of mRNA platform for TBV.

Aotus nancymaae model predicts human immune response to the placental malaria vaccine candidate VAR2CSA.

Placental malaria vaccines (PMVs) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by enzyme-linked immunosorbent assay. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA adhesion of only homologous parasites and not of heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical downselection of PMV candidates and assessment of antibody boosting by PM episodes.

Protein-protein conjugation enhances the immunogenicity of SARS-CoV-2 receptor-binding domain (RBD) vaccines.

Several effective SARS-CoV-2 vaccines have been developed using different technologies. Although these vaccines target the isolates collected early in the pandemic, many have protected against serious illness from newer variants. Nevertheless, efficacy has diminished against successive variants and the need for effective and affordable vaccines persists especially in the developing world. Here, we adapted our protein-protein conjugate vaccine technology to generate a vaccine based on receptor-binding domain (RBD) antigen. RBD was conjugated to a carrier protein, EcoCRM®, to generate two types of conjugates: crosslinked and radial conjugates. In the crosslinked conjugate, antigen and carrier are chemically crosslinked; in the radial conjugate, the antigen is conjugated to the carrier by site-specific conjugation. With AS01 adjuvant, both conjugates showed enhanced immunogenicity in mice compared to RBD, with a Th1 bias. In hACE2 binding inhibition and pseudovirus neutralization assays, sera from mice vaccinated with the radial conjugate demonstrated strong functional activity.

Malaria transmission-blocking conjugate vaccine in ALFQ adjuvant induces durable functional immune responses in rhesus macaques.

Malaria transmission-blocking vaccines candidates based on Pfs25 and Pfs230 have advanced to clinical studies. Exoprotein A (EPA) conjugate of Pfs25 in Alhydrogel® developed functional immunity in humans, with limited durability. Pfs230 conjugated to EPA (Pfs230D1-EPA) with liposomal adjuvant AS01 is currently in clinical trials in Mali. Studies with these conjugates revealed that non-human primates are better than mice to recapitulate the human immunogenicity and functional activity. Here, we evaluated the effect of ALFQ, a liposomal adjuvant consisting of TLR4 agonist and QS21, on the immunogenicity of Pfs25-EPA and Pfs230D1-EPA in Rhesus macaques. Both conjugates generated strong antibody responses and functional activity after two vaccinations though activity declined rapidly. A third vaccination of Pfs230D1-EPA induced functional activity lasting at least 9 months. Antibody avidity increased with each vaccination and correlated strongly with functional activity. IgG subclass analysis showed induction of Th1 and Th2 subclass antibody levels that correlated with activity.

Pfs230 yields higher malaria transmission-blocking vaccine activity than Pfs25 in humans but not mice.

BACKGROUNDVaccines that block human-to-mosquito Plasmodium transmission are needed for malaria eradication, and clinical trials have targeted zygote antigen Pfs25 for decades. We reported that a Pfs25 protein-protein conjugate vaccine formulated in alum adjuvant induced serum functional activity in both US and Malian adults. However, antibody levels declined rapidly, and transmission-reducing activity required 4 vaccine doses. Functional immunogenicity and durability must be improved before advancing transmission-blocking vaccines further in clinical development. We hypothesized that the prefertilization protein Pfs230 alone or in combination with Pfs25 would improve functional activity.METHODSTransmission-blocking vaccine candidates based on gamete antigen Pfs230 or Pfs25 were conjugated with Exoprotein A, formulated in Alhydrogel, and administered to mice, rhesus macaques, and humans. Antibody levels were measured by ELISA and transmission-reducing activity was assessed by the standard membrane feeding assay.RESULTSPfs25-EPA/Alhydrogel and Pfs230D1-EPA/Alhydrogel induced similar serum functional activity in mice, but Pfs230D1-EPA induced significantly greater activity in rhesus monkeys that was enhanced by complement. In US adults, 2 vaccine doses induced complement-dependent activity in 4 of 5 Pfs230D1-EPA/Alhydrogel recipients but no significant activity in 5 Pfs25-EPA recipients, and combination with Pfs25-EPA did not increase activity over Pfs230D1-EPA alone.CONCLUSIONThe complement-dependent functional immunogenicity of Pfs230D1-EPA represents a significant improvement over Pfs25-EPA in this comparative study. The rhesus model is more predictive of the functional human immune response to Pfs230D1 than is the mouse model.TRIAL REGISTRATIONClinicalTrials.gov NCT02334462.FUNDINGIntramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Comparison of carrier proteins to conjugate malaria transmission blocking vaccine antigens, Pfs25 and Pfs230.

Malaria transmission blocking vaccines (TBV) target the sexual stage of the parasite and have been pursued as a stand-alone vaccine or for combination with pre-erythrocytic or blood stage vaccines. Our efforts to develop TBV focus primarily on two antigens, Pfs25 and Pfs230. Chemical conjugation of these poorly immunogenic antigens to carrier proteins enhances their immunogenicity, and conjugates of these antigens to Exoprotein A (EPA) are currently under evaluation in clinical trials. Nonetheless, more potent carriers may augment the immunogenicity of these antigens for a more efficacious vaccine; here, we evaluate a series of proteins to identify such a carrier. Pfs25 and Pfs230 were chemically conjugated to 4 different carriers [tetanus toxoid (TT), a recombinant fragment of tetanus toxin heavy chain (rTThc), recombinant CRM197 produced in Pseudomonas fluorescens (CRM197) or in E. coli (EcoCRM®)] and compared to EPA conjugates in mouse immunogenicity studies. Conjugates of each antigen formulated in Alhydrogel® elicited similar antibody titers but showed differences in functional activity. At a 0.5 µg dose, Pfs230 conjugated to TT, CRM197 and EcoCRM® showed significantly higher functional activity compared to EPA. When formulated with the more potent adjuvant GLA-LSQ, all 4 alternate conjugates induced higher antibody titers as well as increased functional activity compared to the EPA conjugate. IgG subclass analysis of Pfs230 conjugates showed no carrier-dependent differences in the IgG profile. While Alhydrogel® formulations induced a Th2 dominant immune response, GLA-LSQ formulations induced a mixed Th1/Th2 response.

Outer membrane protein complex as a carrier for malaria transmission blocking antigen Pfs230.

Malaria transmission blocking vaccines (TBV) target the mosquito stage of parasite development by passive immunization of mosquitoes feeding on a vaccinated human. Through uptake of vaccine-induced antibodies in a blood meal, mosquito infection is halted and hence transmission to another human host is blocked. Pfs230 is a gametocyte and gamete surface antigen currently under clinical evaluation as a TBV candidate. We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230. We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos® at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines.

Physical-chemical measurement method development for self-assembled, core-shell nanoparticles.

Improvements in dimensional metrology and innovations in physical-chemical characterization of functionalized nanoparticles are critically important for the realization of enhanced performance and benefits of nanomaterials. Toward this goal, we propose a multi-technique measurement approach, in which correlated atomic force microscopy, dynamic light scattering, high performance liquid chromatography and mass spectroscopy measurements are used to assess molecular and structural properties of self-assembled polyplex nanoparticles with a core-shell structure. In this approach, measurement methods are first validated with a model system consisting of gold nanoparticles functionalized with synthetic polycationic branched polyethylenimine macromolecules. Shell thickness is measured by atomic force microscopy and dynamic light scattering, and the polyelectrolyte uptake determined by chromatographic separation and mass spectrometric analysis. Statistical correlation between size, structure and stability provide a basis for extending the methods to more complex self-assembly of nucleic acids and macromolecules via a condensation reaction. From these size and analytical chemical measurements, we obtain a comprehensive spatial description of these assemblies, obtain a detailed interpretation of the core-shell evolution, and identify regions of the parameter space where stable, discrete particle formation occurs.

Development of a bivalent conjugate vaccine candidate against malaria transmission and typhoid fever.

Immune responses to poorly immunogenic antigens, such as polysaccharides, can be enhanced by conjugation to carriers. Our previous studies indicate that conjugation to Vi polysaccharide of Salmonella Typhi may also enhance immunogenicity of some protein carriers. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Pfs25 conjugation to Vi modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against Vi antigen. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever.

Protein-protein conjugate nanoparticles for malaria antigen delivery and enhanced immunogenicity.

Chemical conjugation of polysaccharide to carrier proteins has been a successful strategy to generate potent vaccines against bacterial pathogens. We developed a similar approach for poorly immunogenic malaria protein antigens. Our lead candidates in clinical trials are the malaria transmission blocking vaccine antigens, Pfs25 and Pfs230D1, individually conjugated to the carrier protein Exoprotein A (EPA) through thioether chemistry. These conjugates form nanoparticles that show enhanced immunogenicity compared to unconjugated antigens. In this study, we examined the broad applicability of this technology as a vaccine development platform, by comparing the immunogenicity of conjugates prepared by four different chemistries using different malaria antigens (PfCSP, Pfs25 and Pfs230D1), and carriers such as EPA, TT and CRM197. Several conjugates were synthesized using thioether, amide, ADH and glutaraldehyde chemistries, characterized for average molecular weight and molecular weight distribution, and evaluated in mice for humoral immunogenicity. Conjugates made with the different chemistries, or with different carriers, showed no significant difference in immunogenicity towards the conjugated antigens. Since particle size can influence immunogenicity, we tested conjugates with different average size in the range of 16-73 nm diameter, and observed greater immunogenicity of smaller particles, with significant differences between 16 and 73 nm particles. These results demonstrate the multiple options with respect to carriers and chemistries that are available for protein-protein conjugate vaccine development.

Adjuvant and carrier protein-dependent T-cell priming promotes a robust antibody response against the Plasmodium falciparum Pfs25 vaccine candidate.

Humoral immune responses have the potential to maintain protective antibody levels for years due to the immunoglobulin-secreting activity of long-lived plasma cells (LLPCs). However, many subunit vaccines under development fail to generate robust LLPC responses, and therefore a variety of strategies are being employed to overcome this limitation, including conjugation to carrier proteins and/or formulation with potent adjuvants. Pfs25, an antigen expressed on malaria zygotes and ookinetes, is a leading transmission blocking vaccine (TBV) candidate for Plasmodium falciparum. Currently, the conjugate vaccine Pfs25-EPA/Alhydrogel is in Phase 1 clinical trials in the USA and Africa. Thus far, it has proven to be safe and immunogenic, but it is expected that a more potent formulation will be required to establish antibody titers that persist for several malaria transmission seasons. We sought to determine the contribution of carrier determinants and adjuvants in promoting high-titer, long-lived antibody responses against Pfs25. We found that both adjuvants and carrier proteins influence the magnitude and capacity of Pfs25-specific humoral responses to remain above a protective level. Furthermore, a liposomal adjuvant with QS21 and a TLR4 agonist (GLA-LSQ) was especially effective at inducing T follicular helper (Tfh) and LLPC responses to Pfs25 when coupled to immunogenic carrier proteins.

Increased tumor distribution and expression of histidine-rich plasmid polyplexes.

BACKGROUND: Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays. METHODS: After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo. RESULTS: By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex. CONCLUSIONS: Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer.

Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Enhancement of antifungal activity by integrin-targeting of branched histidine rich peptides.

The treatment of invasive candidiasis associated with growing numbers of immunocompromised patients remains a major challenge complicated by increasing drug resistance. A novel class of branched histidine-lysine (bHK) peptides has promising antifungal activity, and exhibits a mechanism similar to natural histatins, and thus may avoid drug resistance. The present studies evaluate ligand targeting of bHK peptides to fungal surface integrins by determining whether a cyclic RGD (cRGD) peptide with a large PEG linker could enhance bHK peptide antifungal activity. Whereas conjugates containing only the PEG linker reduced bHK peptide activity, conjugates with the cRGD-PEG ligand resulted in marked enhancement of activity against Candida albicans. This study provides the first demonstration of benefit from ligand targeting of antifungal agents to fungal surface receptors.

Surface-modified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition.

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvß3 and αvß5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases.

Buffering capacity and size of siRNA polyplexes influence cytokine levels.

Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production.

Polyethyleneimine (PEI) mediated siRNA gene silencing in the Schistosoma mansoni snail host, Biomphalaria glabrata.

An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a 'mock' fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.

A branched histidine/lysine peptide, H2K4b, in complex with plasmids encoding antitumor proteins inhibits tumor xenografts.

BACKGROUND: In this study we investigated whether a particular branched HK polymer, H2K4b, was an effective in vivo carrier of plasmids expressing the antiangiogenic kringle 1-5 or the tumor suppressor p53. METHODS: H2K4b was synthesized on a solid-phase peptide synthesizer. Distribution, optimization and time course studies were done in tumor-bearing nude mice by systemically administering H2K4b in complex with a luciferase-expressing plasmid. We examined the amount of tumor angiogenesis in C6 with MDA-MB-435 xenografts utilizing the carmine dye. The ability of H2K4b to carry luciferase plasmids to different tissues was compared with several liposomal carriers. Medium from cells transfected with mKr1-5 was tested for its capacity to inhibit angiogenesis with an in vivo Matrigel assay. We then determined if systemically delivered H2K4b in complex with plasmid encoding mKr1-5 inhibited tumor growth; we also compared the antitumor activity of HK polyplexes containing hKr1-5, mKr1-5, and p53 plasmids. RESULTS: H2K4b carried the luciferase-expressing plasmid in order of descending efficacy to these tissues: lung, spleen, tumor, and liver. Compared to DOTAP-containing liposomes, H2K4b was a more effective carrier of a luciferase-containing plasmid to extrapulmonary tissues. We then determined that mKr1-5 in complex with H2K4b reduced MDA-MB-435 tumor growth by approximately 50% compared to the control group (P < 0.01). Similarly, H2K4b/mKr1-5 polyplexes reduced the growth of C6 xenografts. In MDA-MB-435 xenografts, p53- and Kr1-5-expressing plasmids in complex with H2K4b had comparable antitumor activity. CONCLUSION: H2K4b demonstrates potential as a carrier of plasmids encoding antiangiogenic and/or tumor suppressor proteins in a tumor-bearing mouse model.

Transporting silence: design of carriers for siRNA to angiogenic endothelium.

The recently developed siRNA oligonucleotides are an attractive alternative to antisense as a therapeutic modality because of their robust, gene selective silencing of drug target protein expression. To achieve therapeutic success, however, several hurdles must be overcome including rapid clearance, nuclease degradation, and inefficient intracellular localization. In this presentation, we discuss design strategies for development of self-assembling nanoscale carriers for neovasculature targeted delivery of siRNA inhibiting tumor or ocular angiogenesis.

Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis.

OBJECTIVE: RNA interference is a process in which genes can be silenced sequence-specifically. In mammals, RNA interference can be invoked by introduction of small (19-21-nucleotide) double-stranded RNA molecules known as small interfering RNA (siRNA) into cells. Thereby, siRNA offers promise as a novel therapeutic modality. However, siRNA is a relatively large, highly charged molecule and does not readily enter cells. This study was undertaken to investigate the use of electroporation for in vivo transfection of siRNA into joint tissue in arthritic mice to achieve local RNA interference. METHODS: Proof of principle that siRNA is able to inhibit gene expression in vivo in the mouse joint was studied by local injection and electroporation of siRNA designed to silence reporter genes. In mice with collagen-induced arthritis (CIA), the disease-modulating activity of siRNA designed to silence tumor necrosis factor alpha (TNFalpha) was investigated. RESULTS: Luciferase activity could be reduced by >90% with luciferase-specific siRNA as compared with the activity measured after electroporation without siRNA or with irrelevant siRNA. The effect was observed only locally. In mice with CIA, electroporation of siRNA designed to inhibit TNFalpha strongly inhibited joint inflammation, whereas electroporation of irrelevant siRNA or injection of siRNA against TNFalpha without electroporation failed to produce therapeutic effects. CONCLUSION: Local electroporation of siRNA in joint tissue can inhibit CIA in mice. These results offer promise for the use of siRNA as a new strategy for therapeutic intervention in rheumatoid arthritis and may serve as a tool to study arthritis disease pathways through loss-of-function phenotypes.