Therapeutic Cassette Research Articles

Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5' or 3'-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.

Systemic Correction of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8(+) T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8(+) CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Gene Specificity of Suppression of Transgene-Mediated Insertional Transcriptional Activation by the Chicken HS4 Insulator

Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value.

Semi-mechanistic pharmacodynamic modelling of gene expression and silencing processes

Background Suppressed expression of transgene is a major obstacle in gene therapy. Understanding of the mechanisms involved in expression and silencing of exogenous genes is required to overcome gene therapy hurdles. Purpose To develop a semi-mechanistic model describing the effects of transgenes over the activity of an expression cassette. Methods Twelve Balb/c mice received 40 μg of plasmid DNA. Animals were assigned to one of the following treatments: (I) 20 μg of the plasmid expressing luciferase (pEF-Luc) and 20 μg of “empty” plasmid; (II) pEF-Luc (20 μg) and 20 μg of plasmid expressing murine interferon alpha (IFNα); and (III) pEF-Luc (20 μg), and 20 μg of plasmid expressing β-galactosidase (pCMVβ). The expression of luciferase over time, quantified by a noninvasive method, was used as a measured of pEF-Luc activity and modelled using NONMEM. Results The selected model suggests the co-existence of two forms of active DNA differing in their transcription efficiencies. The core model was expanded to describe reversible and irreversible silencing processes, induced by the coexpression of IFNα or β-galactosidase, respectively. Conclusion Coupling noninvasive in vivo imaging and mathematical modelling allows quantitative description of gene transfer, providing a tool to select the best regulatory elements to construct a therapeutic expression cassette.

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.

A highly functional mini-dystrophin / GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy

A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.7 kb miniDys-GFP fusion gene by replacing the dystrophin C-terminal domain (DeltaCT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (DeltaH2-R19). In a transgenic mdx(4Cv) mouse expressing the miniDys-GFP fusion protein under the control of a skeletal muscle-specific promoter, the green fusion protein localized on the sarcolemma, where it assembled the dystrophin-glycoprotein complex and completely prevented the development of dystrophy in transgenic mdx(4Cv) muscles. When myogenic and other stem cells from these mice were transplanted into mdx(4Cv) recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx(4Cv) mice reconstituted with bone marrow cells from the transgenic mice, we monitored engraftment in various muscle groups and found the number of miniDys-GFP(+) fibers increased with time. We suggest that these transgenic mdx(4Cv) mice are highly useful for developing autologous cell therapies for DMD.

464. Development of a Novel Gene Regulation System Downstream of Promoter

Current regulation systems for vector delivered transgene has focused primarily on controlling promoter activity. In this study, we assayed the ability to regulate expression of transgene cassettes after vector delivery downstream of promoter activity through use of alternative splicing. A unique human beta globin intron carrying a point mutation causing aberrant splicing was engineered into the open reading frame of vector expressing reporter or therapeutic transgene cassettes under constitutively expressing CBA promoter. After introduction into target cells, vector derived mRNAs are aberrantly spliced and no functional proteins are produced. Administration of target antisense oligonucleotides (AON) specific for alternative splice site restores wild type splicing and functional protein expression. Using this approach, we transfected luciferase reporter transgene cassettes carrying IVS2-654 intron plasmids into 293 cells. 24 hr post transfection cell lysates were obtained and luciferase assays preformed. We observed 6-8 folds increase in luciferase activity after AON treatment equivalent to levels obtained with wile type intron. This restoration of correct splicing and protein expression was not dependent on location of IVS2-654 intron. Regulated expression in vivo was tested using AAV vector carry regulated transgene cassette in mouse model. One week after portal vein injection of 1010 genomes, 25mg/kg AON was administered IP. We observed an 8-10 folds increase over control that persisted for over 1 month before returning to base-line level. Long-term study shows AON regulation 1yr. post vector administration. To further optimize this regulation system, we constructed a series of intron mutants that removed 250-400nt of 5' sequences as well as engineered point mutations that would strengthen use of alternative splice sites. Results of these efforts resulted in mutant intron cassettes that reduced background expression dramatically and resulted in 160-230 folds increase in protein expression after oligo treatment.

1102. Stable, Non-Viral Gene Transfer to Human CD34+ Hematopoietic Progenitor Cells Using the Sleeping Beauty Transposon

Primary hematopoietic stem cells (HSC), capable of repopulating the entire hematopoietic and immune system with the full complement of cell types, are an attractive target for gene therapy strategies to treat diseases of blood cells. Gene modification of a patient's HSC could be used for autologous transplantation to treat inherited genetic disorders, including Severe Combined Immune Deficiency (SCID) and other immune deficiencies. Non-viral strategies have become available for gene transfer into cells that result in integration of therapeutic gene cassettes. For example, Sleeping Beauty (SB) transposes directly from one DNA locus to another through the activity of the transposase enzyme recognizing and performing excision and integration of the DNA sequences between specific inverted repeat (IR) sequences of the transposon. Transposase-mediated integration of the IR-flanked expression cassette into chromosomes provides the basis for long-term transgene expression. Here we used the transposon-based gene delivery strategy composed of a two plasmid system: a plasmid containing the SB transposase gene (for transient production of the transposase), and a second plasmid containing an expression cassette with a reporter gene flanked by the SB IR (inverted repeat) sequences. For this study, we evaluated the potential use of the SB transposon system to deliver genes in a stable manner to human hematopoietic cells by electroporation using the Amaxa nucleoporator. One plasmid carrying an enhanced Green Fluorescent Protein (eGFP) reporter cassette (to facilitate FACS analysis of resultant cells) flanked by the SB IR sequences for permanent integration into the target cell chromosomes was co-nucleoporated with a second plasmid carrying the SB transposase expression cassette. We first used the K562 human erythro-leukemia cell line to optimize several parameters of gene delivery, achieving high levels (50-80%) of stable gene integration with optimal amounts and ratios of the two plasmids. Southern analysis of K562 clonal populations showed that 1 to 6 copies of the transposon can be detected. We then nucleoporated the SB-based system into human CD34+ progenitor cells isolated from umbilical cord blood and achieved initial gene transfer to these primary cells at moderate levels (10-30%) that led to stable expression of the reporter gene (1-4%). Results from these studies demonstrate the potential for using the SB transposon system for gene therapy using HSC.

Improved Production and Purification of Minicircle DNA Vector Free of Plasmid Bacterial Sequences and Capable of Persistent Transgene ExpressionIn Vivo

We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.

141. Development of a One-Step Column Purification of Minicircle Vector DNA Devoid of Bacterial Sequences That Results in High Level Persistent Transgene Expression In Vivo

We have shown that episomal circular DNA vectors free of plasmid bacterial DNA sequences are capable of indefinite high level transgene expression in vivo. The minicircle vector is generated in bacteria from a parental plasmid containing an inducible phiC31 integrase gene, and att P and att B recognition sites flanking the therapeutic expression cassette. Recombination results in the formation of two circular DNA molecules, one containing the plasmid bacterial backbone and the other the eukaryotic expression cassette. Previously, the minicircle was purified away from the plasmid bacterial DNA by cesium chloride banding in an ultracentrifuge (Chen et al., Mol Ther. 8:495, 2003). We have now devised a one-step purification method that results in a high yield of minicircle vector. To do this, two copies of the phiC31 integrase gene under the control of an inducible promoter (BAD), together with the transgene expression cassette flanked by the attB and attP sites, were built into a minicircle-producing plasmid, p2phiC31. An intron encoded restriction enzyme I-Sce I gene, also under the control of the BAD promoter, together with an I-Sce I restriction site, were also built into p2phiC31. Top 10 E. coli was used to amplify p2phiC31 using standard plasmid preparation. To induce the phiC31 integrase-mediated minicircle formation, an over night culture was re-suspended 4 to 1 (vol/vol) in fresh LB broth containing the pBAD inducer 1% L-arabinose and incubated at 32°C with constant shaking for one hour. The temperature of the incubation was increased to 37°C, to optimize I-Sce I enzymatic activity, for an additional 2 hours. Linearization of the plasmid bacterial DNA circle by the restriction enzyme resulted in its rapid degradation by exonucleases in bacteria. The mini-circle expression cassette becomes the only episomal circular DNA and is easily isolated by an affinity purification column. Using this technology to prepare a minicircle encoding a 4 kb human factor IX expression cassette, > 1 mg of minicircle of 97 percent purity can be prepared from 1 liter of over night bacterial growth. When injected into the livers of animals by hydrodynamic infusion, about 10 μg/ml of serum hFIX was expressed for up to 7 weeks (length of study). The high yield, simple purification, and robust and persistence of transgene expression makes these vectors viable for in vivo gene therapy applications.

718. Systemic Gene Transfer to Striated Muscles Using rAAV6 Vectors

Human mortality is severely affected by diseases of the cardiac and skeletal musculature. Genetic interventions are being developed for these diseases, but are limited by an inability to achieve widespread gene transfer to the heart and numerous skeletal muscles of an adult mammal. We have observed that recombinant adeno-associated viral vectors (rAAV vectors) comprising serotype type 6 capsid proteins potently transduce striated muscles following direct intramuscular injection. In subsequent experiments we have identified procedures for administering rAAV6 vectors intravascularly to conscious adult mice that achieve high-level transgene expression (up to 8,000 fold increases in indices over control values) in the vast majority of cardiac and skeletal muscle fibers. These data are the first to demonstrate that extensive transduction of both the cardiac and skeletal musculature is achievable in an adult mammal using a single, minimally invasive intravenous injection of rAAV6 vectors. We have observed that transgene expression in striated muscle fibers following vector administration is influenced by the design of the expression cassette delivered and present data demonstrating that muscle-restricted transgene expression reduces the incidence of immunological reaction against the transgene product compared with a constitutively-driven expression cassette. To assess the therapeutic potential of these novel gene delivery techniques, we have begun administering rAAV6 vectors containing therapeutic expression cassettes to murine models of Duchenne muscular dystrophy. Treated dystrophic mdx mice exhibit widespread expression of therapeutic dystrophin-based proteins throughout the striated musculature in a manner that is sufficient to reduce pathological features of the dystrophic phenotype, including susceptibility to contraction induced injury, and serum creatine kinase (an indicator of muscle degeneration). Having established a method of facilitating widespread gene delivery throughout the muscles of adult mice, we are now evaluating modifications of these techniques for increased gene delivery efficiency. Here we summarize our understanding of the factors influencing rAAV-mediated systemic gene delivery to striated muscles, and the therapeutic potential of such techniques for the treatment of muscular dystrophies.

Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-kappaB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6kappaB-EDL1E-tk, containing six copies of the NF-kappaB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6kappaB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6kappaB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6kappaB-EDL1E-tk or GCV alone. Similarly, no retardation of tumor growth was observed in mice containing LMP1-negative CT-26 tumors injected with both the p6kappaB-EDL1E-tk cassette and GCV. We propose that intratumoral injection of therapeutic agents, such as DNA of transcription-regulated cassette and GCV, via in vivo electroporation may be used as an alternative treatment for EBV LMP1-expressing cancers.

Multiple advantages of alpha-fetoprotein as a marker for in vivo gene transfer.

The development of improved gene transfer vectors has been hampered by the lack of a nonimmunogenic reporter gene that can be serially quantified in the serum or from other sites. In response to the need to develop a new reporter protein, we have evaluated alpha-fetoprotein (AFP) as a potential candidate. A first-generation E1/E3-deleted adenoviral vector expressing human AFP (hAFP) was generated as a preliminary tool to evaluate AFP as a reporter. Using both mouse and baboon models, hAFP expression was evaluated in serum after intravenous delivery and in serum and bronchioalveolar lavage (BAL) fluid after delivery to the lung. In immunocompetent animals, intravenous delivery of the hAFP adenoviral vector resulted in hAFP expression in the serum early after injection, which declined rapidly over time. Disappearance of hAFP from the serum was complete by 3-4 weeks after administration and was accompanied by robust antibody responses to hAFP and loss of infected cells. After lung delivery, hAFP could be detected in both serum and BAL. This allowed the analysis of the kinetics of gene expression in the lung without sacrificing the animals. In both liver and lung, immunohistochemical analysis correlated well with hAFP levels as detected in serum or BAL, indicating that serum levels were a reliable marker of tissue expression. Preliminary results with a mouse AFP expressed in a helper-dependent adenoviral vector indicate that use of a species-specific version of AFP will eliminate the complication of antibody development. These initial evaluations suggest that AFP is useful as a reporter gene to evaluate gene expression of therapeutic cassettes in multiple tissues, and it should be considered for use in human subjects.

Genetic Prodrug Activation Therapy for Breast Cancer: A Phase I Clinical Trial oferbB-2–Directed Suicide Gene Expression

This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2-overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2-positive tumor cells, confirming the selectivity of the approach. The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.