Prospects of viral vector-mediated delivery of sequences encoding anti-HBV designer endonucleases.

Available treatment for chronic hepatitis B virus (HBV) infection offers modest functional curative efficacy. The viral replicative intermediate comprising covalently closed circular DNA (cccDNA) is responsible for persistent chronic HBV infection. Hence, current efforts have focused on developing therapies that disable cccDNA. Employing gene editing tools has emerged as an attractive strategy, with the end goal of establishing permanently inactivated cccDNA. Although anti-HBV designer nucleases are effective in vivo, none has yet progressed to clinical trial. Lack of safe and efficient delivery systems remains the limiting factor. Several vectors may be used to deliver anti-HBV gene editor-encoding sequences, with viral vectors being at the forefront. Despite the challenges associated with packaging large gene editor-encoding sequences into viral vectors, advancement in the field is overcoming such limitations. Translation of viral vector-mediated gene editing against HBV to clinical application is within reach. This review discusses the prospects of delivering HBV targeted designer nucleases using viral vectors.

Self-amplifying RNA vaccines for infectious diseases.

Vaccinology is shifting toward synthetic RNA platforms which allow for rapid, scalable, and cell-free manufacturing of prophylactic and therapeutic vaccines. The simple development pipeline is based on in vitro transcription of antigen-encoding sequences or immunotherapies as synthetic RNA transcripts, which are then formulated for delivery. This approach may enable a quicker response to emerging disease outbreaks, as is evident from the swift pursuit of RNA vaccine candidates for the global SARS-CoV-2 pandemic. Both conventional and self-amplifying RNAs have shown protective immunization in preclinical studies against multiple infectious diseases including influenza, RSV, Rabies, Ebola, and HIV-1. Self-amplifying RNAs have shown enhanced antigen expression at lower doses compared to conventional mRNA, suggesting this technology may improve immunization. This review will explore how self-amplifying RNAs are emerging as important vaccine candidates for infectious diseases, the advantages of synthetic manufacturing approaches, and their potential for preventing and treating chronic infections.

Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells.

Chimeric antigen receptor (CAR) T cell technology has enabled successfully novel concepts to treat cancer patients, with substantial remission rates in lymphoid malignancies. This cell therapy is based on autologous T lymphocytes that are genetically modified to express a CAR that recognizes tumor-associated antigens and mediates the elimination of the respective tumor cells. Current limitations include laborious manufacturing procedures as well as severe immunological side effects upon administration of CAR T cells. To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus. Using CRISPR-Cas9 and adeno-associated virus-based donor vectors, some 60% of genome-edited T cells were CAR+/CD20+/CD34+/CD52- without further selection. This could be increased to >95% purity after CD34 tag-based positive selection. These epitope-switched CAR T cells retained cell killing competence against CD19+ tumor cells, and were resistant to alemtuzumab (anti-CD52) but sensitive to rituximab (anti-CD20) in complement-dependent cytotoxicity assays. In conclusion, gene editing-based multiple epitope switching represents a promising development with the potential to improve both the manufacturing procedure as well as the clinical safety of CAR T cells.

Advances in designing Adeno-associated viral vectors for development of anti-HBV gene therapeutics.

Despite the five decades having passed since discovery of the hepatitis B virus (HBV), together with development of an effective anti-HBV vaccine, infection with the virus remains a serious public health problem and results in nearly 900,000 annual deaths worldwide. Current therapies do not eliminate the virus and viral replication typically reactivates after treatment withdrawal. Hence, current endeavours are aimed at developing novel therapies to achieve a functional cure. Nucleic acid-based therapeutic approaches are promising, with several candidates showing excellent potencies in preclinical and early stages of clinical development. However, this class of therapeutics is yet to become part of standard anti-HBV treatment regimens. Obstacles delaying development of gene-based therapies include lack of clinically relevant delivery methods and a paucity of good animal models for preclinical characterisation. Recent studies have demonstrated safety and efficiency of Adeno-associated viral vectors (AAVs) in gene therapy. However, AAVs do have flaws and this has prompted research aimed at improving design of novel and artificially synthesised AAVs. Main goals are to improve liver transduction efficiencies and avoiding immune clearance. Application of AAVs to model HBV replication in vivo is also useful for characterising anti-HBV gene therapeutics. This review summarises recent advances in AAV engineering and their contributions to progress with anti-HBV gene therapy development.

Inhibition of replication of hepatitis B virus using transcriptional repressors that target the viral DNA.

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS: To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS: Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS: Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.

Progress With Developing Use of Gene Editing To Cure Chronic Infection With Hepatitis B Virus.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 6% of the world's population. Carriers of the virus are at risk for life-threatening complications, and developing curative treatment remains a priority. The main shortcoming of licensed therapies is that they do not affect viral covalently closed circular DNA (cccDNA), a stable intermediate of replication. Harnessing gene editing to mutate cccDNA provides the means to inactivate HBV gene expression permanently. Reports have described use of engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) nucleases. Although inhibition of viral replication has been demonstrated, reliably detecting mutations in cccDNA has been difficult. Also, the dearth of murine models that mimic cccDNA formation has hampered analysis in vivo. To reach a stage of clinical use, efficient delivery of the editors to HBV-infected hepatocytes and limiting unintended off-target effects will be important. Investigating therapeutic efficacy in combination with other treatment strategies, such as immunotherapies, may be useful to augment antiviral effects. Advancing gene editing as a mode of treating HBV infection is now at an interesting stage and significant progress is likely to be made in the immediate future.

Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus.

Chronic infection with hepatitis B virus (HBV) remains an important global health problem. Currently licensed therapies have modest curative efficacy, which is as a result of their transient effects and limited action on the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Gene editing with artificial HBV-specific endonucleases and use of artificial activators of the RNA interference pathway have shown anti-HBV therapeutic promise. Although results from these gene therapies are encouraging, maximizing durable antiviral effects is important. To address this goal, a strategy that entails combining gene editing with homology-directed DNA recombination (HDR), to introduce HBV-silencing artificial primary microRNAs (pri-miRs) into HBV DNA targets, is reported here. Previously described transcription activator-like effector nucleases (TALENs) that target the core and surface sequences of HBV were used to introduce double stranded breaks in the viral DNA. Simultaneous administration of donor sequences encoding artificial promoterless anti-HBV pri-miRs, with flanking arms that were homologous to sequences adjoining the TALENs' targets, augmented antiviral efficacy. Analysis showed targeted integration and the length of the flanking homologous arms of donor DNA had a minimal effect on antiviral efficiency. These results support the notion that gene editing and silencing may be combined to effect improved inhibition of HBV gene expression.

Stealth lipoplex decorated with triazole-tethered galactosyl moieties: a strong hepatotropic gene vector.

Mono-antennary galacto derivatives of cholesterol are being actively developed to direct lipoplexes to the asialoglycoprotein receptor (ASGP-R) on hepatocytes. Here we report on a novel ASGP-R ligand cholest-5-en-3-yl [1-(ß-D-galactopyranosyl)-1H-1,2,3-triazol-4-yl]methylcarbamate (4), assembled by a copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry), and compare it with cholest-5-en-3-yl-ß-D-galactopyranoside (2) and cholest-5-en-3-yl [1-(ß-D-galactopyranosyl-1'-oxy)phen-4-yl]carbamate (3), in liposome formulations with or without 5 mol% distearoylphosphatidylethanolamine poly(ethylene glycol)2000, intended for DNA delivery to ASGP-R-positive hepatocyte-derived HepG2 cells and the ASGP-R-negative embryo kidney cell line HEK293. Transfection levels attained with lipoplex 4 were 100 and 300% greater than those for lipoplexes 2 and 3 respectively in HepG2 cells, while competition assays reduced transfection levels by up to 98%. Transfection activities achieved in HEK293 cells were up to three orders of magnitude lower. Therefore, 4 is representative of a new class of promising hepatotropic ligands for gene delivery.

Recent advances in use of gene therapy to treat hepatitis B virus infection.

Chronic infection with hepatitis B virus (HBV) occurs in approximately 5 % of the world's human population and persistence of the virus is associated with serious complications of cirrhosis and liver cancer. Currently available treatments are modestly effective and advancing novel therapeutic strategies is a medical priority. Stability of the viral cccDNA replication intermediate is a major factor that has impeded the development of therapies that are capable of eliminating chronic infection. Recent advances that employ gene therapy strategies offer useful advantages over current therapeutics. Silencing of HBV gene expression by harnessing the RNA interference pathway has been shown to be highly effective in cell culture and in vivo. However, a potential limitation of this approach is that the post-transcriptional mechanism of gene silencing does not disable cccDNA. Early results using designer transcription activator-like effector nucleases (TALENs) and repressor TALEs (rTALEs) have shown potential as a mode of inactivating cccDNA. In this article, we review the recent advances that have been made in HBV gene therapy, with a particular emphasis on the potential anti-HBV therapeutic utility of designed sequence-specific DNA binding proteins and their derivatives.

Progress and Prospects of Anti-HBV Gene Therapy Development.

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV.

Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases.

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.

Inhibition of hepatitis B virus replication in cultured cells and in vivo using 2'-O-guanidinopropyl modified siRNAs.

Silencing hepatitis B virus (HBV) gene expression with exogenous activators of the RNA interference (RNAi) pathway has shown promise as a new mode of treating infection with the virus. However, optimizing efficacy, specificity, pharmacokinetics and stability of RNAi activators remains a priority before clinical application of this promising therapeutic approach is realised. Chemical modification of synthetic short interfering RNAs (siRNAs) provides the means to address these goals. This study aimed to assess the benefits of incorporating nucleotides with 2'-O-guanidinopropyl (GP) modifications into siRNAs that target HBV. Single GP residues were incorporated at nucleotide positions from 2 to 21 of the antisense strand of a previously characterised effective antiHBV siRNA. When tested in cultured cells, siRNAs with GP moieties at selected positions improved silencing efficacy. Stability of chemically modified siRNAs in 80% serum was moderately improved and better silencing effects were observed without evidence for toxicity or induction of an interferon response. Moreover, partially complementary target sequences were less susceptible to silencing by siRNAs with GP residues located in the seed region. Hydrodynamic co-injection of siRNAs with a replication-competent HBV plasmid resulted in highly effective knock down of markers of viral replication in mice. Evidence for improved efficacy, reduced off target effects and good silencing in vivo indicate that GP-modifications of siRNAs may be used to enhance their therapeutic utility.

Production, Characterization, and Assessment of Permanently Cationic and Ionizable Lipid Nanoparticles for Use in the Delivery of Self-Amplifying RNA Vaccines.

Africa bears the highest burden of infectious diseases, yet the continent is heavily reliant on First World countries for the development and supply of life-saving vaccines. The COVID-19 pandemic was a stark reminder of Africa's vaccine dependence and since then great interest has been generated in establishing mRNA vaccine manufacturing capabilities on the African continent. Herein, we explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as an alternative to the conventional mRNA vaccine platform. The approach is intended to produce dose-sparing vaccines which could assist resource-constrained countries to achieve vaccine independence. Protocols to synthesize high-quality saRNAs were optimized and in vitro expression of reporter proteins encoded by saRNAs was achieved at low doses and observed for an extended period. Permanently cationic or ionizable LNPs (cLNPs and iLNPs, respectively) were successfully produced, incorporating saRNAs either exteriorly (saRNA-Ext-LNPs) or interiorly (saRNA-Int-LNPs). DOTAP and DOTMA saRNA-Ext-cLNPs performed best and were generally below 200 nm with good PDIs (<0.3). DOTAP and DDA saRNA-Int-cLNPs performed optimally, allowing for saRNA amplification. These were slightly larger, with higher PDIs as a result of the method used, which will require further optimization. In both cases, the N:P ratio and lipid molar ratio had a distinct effect on saRNA expression kinetics, and RNA was encapsulated at high percentages of >90%. These LNPs allow the delivery of saRNA with no significant toxicity. The optimization of saRNA production and identification of potential LNP candidates will facilitate saRNA vaccine and therapeutic development. The dose-sparing properties, versatility, and manufacturing simplicity of the saRNA platform will facilitate a rapid response to future pandemics.

AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy.

Hepatitis B virus (HBV) has afflicted humankind for decades and there is still no treatment that can clear the infection. The development of recombinant adeno-associated virus (rAAV)-based gene therapy for HBV infection has become important in recent years and research has made exciting leaps. Initial studies, mainly using mouse models, showed that rAAVs are non-toxic and induce minimal immune responses. However, several later studies demonstrated rAAV toxicity, which is inextricably associated with immunogenicity. This is a major setback for the progression of rAAV-based therapies toward clinical application. Research aimed at understanding the mechanisms behind rAAV immunity and toxicity has contributed significantly to the inception of approaches to overcoming these challenges. The target tissue, the features of the vector, and the vector dose are some of the determinants of AAV toxicity, with the latter being associated with the most severe adverse events. This review discusses our current understanding of rAAV immunogenicity, toxicity, and approaches to overcoming these hurdles. How this information and current knowledge about HBV biology and immunity can be harnessed in the efforts to design safe and effective anti-HBV rAAVs is discussed.

Opportunities and challenges of leveraging COVID-19 vaccine innovation and technologies for developing sustainable vaccine manufacturing capabilities in Africa.

The COVID-19 pandemic heralded unprecedented resource mobilisation and global scientific collaboration to rapidly develop effective vaccines. Regrettably, vaccine distribution has been inequitable, particularly in Africa where manufacturing capacity remains nominal. To address this, several initiatives are underway to develop and manufacture COVID-19 vaccines in Africa. Nevertheless, diminishing demand for COVID-19 vaccines, the cost competitiveness of producing goods locally, intellectual property rights issues, and complex regulatory environments among other challenges can undermine these ventures. We outline how extending COVID-19 vaccine manufacturing in Africa to include diverse products, multiple vaccine platforms, and advanced delivery systems will ensure sustainability. Possible models, including leveraging public-academic-private partnerships to enhance success of vaccine manufacturing capacity in Africa are also discussed. Intensifying research in vaccine discovery on the continent could yield vaccines that further bolster sustainability of local production, ensuring greater pandemic preparedness in resource-constrained environments, and long-term health systems security.

MicroRNA-like antivirals.

Employing engineered DNA templates to express antiviral microRNA (miRNA) sequences has considerable therapeutic potential. The durable silencing that may be achieved with these RNAi activators is valuable to counter chronic viral infections, such as those caused by HIV-1, hepatitis B, hepatitis C and dengue viruses. Early use of expressed antiviral miRNAs entailed generation of cassettes containing Pol III promoters (e.g. U6 and H1) that transcribe virus-targeting short hairpin RNA mimics of precursor miRNAs. Virus escape from single gene silencing elements prompted later development of combinatorial antiviral miRNA expression cassettes that form multitargeting siRNAs from transcribed long hairpin RNA and polycistronic primary miRNA sequences. Weaker Pol III and Pol II promoters have also been employed to control production of antiviral miRNA mimics, improve dose regulation and address concerns about toxicity caused by saturation of the endogenous miRNA pathway. Efficient delivery of expressed antiviral sequences remains challenging and utilizing viral vectors, which include recombinant adenoviruses, adeno-associated viruses and lentiviruses, has been favored. Investigations using recombinant lentiviruses to transduce CD34+ hematological precursor cells with expressed HIV-1 gene silencers are at advanced stages and show promise in preclinical and clinical trials. Although the use of expressed antiviral miRNA sequences to treat viral infections is encouraging, eventual therapeutic application will be dependent on rigorously proving their safety, efficient delivery to target tissues and uncomplicated large scale preparation of vector formulations. This article is part of a special issue entitled: MicroRNAs in viral gene regulation.

Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells.

BACKGROUND: Conventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target. RESULTS: shRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels. CONCLUSIONS: This study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.

Countering hepatitis B virus infection using RNAi: how far are we from the clinic?

Globally, persistent HBV infection is a significant cause of public health problems. Currently available HBV therapies have variable efficacy and there is a need to develop improved treatment to prevent cirrhosis and hepatocellular carcinoma. Although RNA interference (RNAi)-based approaches have shown promise, accomplishing safe and sustained silencing by RNAi activators, as well as their efficient delivery to hepatocytes have hampered clinical translation of this very promising technology. Expressed silencers may be produced in a sustained manner from stable DNA templates, which makes them suited to treatment of chronic HBV infection. DNA expression cassettes can be incorporated into both viral and non-viral vectors, but in vivo delivery of these cassettes with non-viral vectors is currently inefficient. Synthetic short interfering RNAs (siRNAs), which may be chemically modified to improve stability, specificity and efficacy, are more conveniently delivered to their cytoplasmic sites of action with synthetic non-viral vectors. However, the short duration of action of this class of RNAi activator is a drawback for treatment of chronic HBV infection. Despite the impressive progress that has been made in developing highly effective HBV gene silencers, challenges continue to face implementation of RNAi-based HBV therapy. This review will discuss the current status of the topic and consider the developments that are required to advance RNAi-based HBV therapy to clinical application.

Synthesis of 2'-O-guanidinopropyl-modified nucleoside phosphoramidites and their incorporation into siRNAs targeting hepatitis B virus.

Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3' ends. To improve efficacy of siRNAs, chemical modification at the 2'-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic. To assess the potential utility of introducing positively charged groups at the 2' position, our investigations aimed at assessing efficacy of novel siRNAs containing 2'-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate antihepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.

Deriving four functional anti-HIV siRNAs from a single Pol III-generated transcript comprising two adjacent long hairpin RNA precursors.

Several different approaches exist to generate expressed RNA interference (RNAi) precursors for multiple target inhibition, a strategy referred to as combinatorial (co)RNAi. One such approach makes use of RNA Pol III-expressed long hairpin RNAs (lhRNAs), which are processed by Dicer to generate multiple unique short interfering siRNA effectors. However, because of inefficient intracellular Dicer processing, lhRNA duplexes have been limited to generating two independent effective siRNA species. In this study, we describe a novel strategy whereby four separate anti-HIV siRNAs were generated from a single RNA Pol III-expressed transcript. Two optimized lhRNAs, each comprising two active anti-HIV siRNAs, were placed in tandem to form a double long hairpin (dlhRNA) expression cassette, which encodes four unique and effective siRNA sequences. Processing of the 3' position lhRNA was more variable but effective multiple processing was possible by manipulating the order of the siRNA-encoding sequences. Importantly, unlike shRNAs, Pol III-expressed dlhRNAs did not compete with endogenous and exogenous microRNAs to disrupt the RNAi pathway. The versatility of expressed lhRNAs is greatly expanded and we provide a mechanism for generating transcripts with modular lhRNAs motifs that contribute to improved coRNAi.