Transgene Expression In Mouse Liver Research Articles

Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle

Adeno-associated virus (AAV) has great potential as a source of treatments for conditions that might respond to potent and ubiquitous transgene expression. However, among its drawbacks, the genetic “payload” of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are sizeable and susceptible to transcriptional silencing. We recently described a short (404 bp), potent, and persistent promoter obtained from the genome of pseudorabies virus (PrV) called alphaherpesvirus latency-associated promoter 2 (LAP2). Here, we evaluated the biodistribution and potency of transgene expression in mouse peripheral tissues in response to local and systemic administration of AAV8-LAP2 and AAV9-LAP2. We found that administration of these vectors resulted in levels of transgene expression that were similar to the larger EF1α promoter. LAP2 drives potent transgene expression in mouse liver and kidney when administered systemically and in skeletal muscle in response to intramuscular delivery. Notably, in skeletal muscle, administration of vectors with LAP2 and EF1α promoters resulted in preferential transduction of myofibers type 2. A direct side-by-side comparison between LAP2 and the EF1α promoter revealed that, despite its smaller size, LAP2 was equally potent to the EF1α promoter and resulted in widespread gene expression after IV and IM administration of AAV8 or AAV9 vectors. Collectively, these findings suggest that constructs that include LAP2 may have the capacity to deliver large therapeutically effective payloads in support of future gene therapy protocols.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

ObjectiveMelittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. MethodsThe melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. ResultsrAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. ConclusionPre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Conventional plasmid DNAs with a CpG-containing backbone achieve durable transgene expression in mouse liver.

Durable transgene expression from plasmid DNAs is the key to gene therapy with non-viral vectors. A comparison of the durability of transgene expression from plasmid DNAs with the CpG-free and -containing backbones is important. We constructed plasmid DNAs with the CpG-containing backbone, various transcription regulatory sequences with and without CpG, and the gene encoding Gaussia princeps luciferase, which is apparently non-immunogenic. The tail vein hydrodynamics-based method was used for plasmid injection into mice, and the luciferase activity in serum was tracked for 28 days. The plasmid DNAs containing the albumin promoter [with or without the cytomegalovirus (CMV) enhancer] and the elongation factor (EF)1α promoter plus the CMV enhancer exhibited long-term luciferase expression. The expression from the plasmid DNA containing the albumin promoter without the CMV enhancer was maintained for at least 24 weeks and was similar to that from the corresponding CpG-free plasmid DNA. The results obtained in the present study suggest that special sequences/systems are unnecessary for durable transgene expression from plasmid DNAs when the proper transcription regulatory sequences are used.

147. Transcription Factor Binding Site in Plasmid Regulates Persistence of Transgene Expression in Mouse Liver

Persistent expression of transgene is most desirable for treatment of genetic diseases. It is primarily controlled by genetic elements in the promoter region where transcription factors bind. The objective of this study is to assess the role of the transcription factor binding sites (TFBS) present in the most commonly used CMV and albumin promoters containing the promoter core sequence and several TFBS for ATF1, NFκB, SRF, AP1, Sp1, NF1α, CEBPA, HNF4α, and HNF1 in determining persistency of transgene expression in mice. Secreted embryonic alkaline phosphatase (SEAP) and interleukin 10 (IL-10) were used as a reporter to monitor transgene expression. Variousdeletion mutation plasmids at the promoter region were made to examine the function of each of the TFBS in regulating reporter gene expression. Similarly, a series of new plasmids containing only one TFBS inserted at the 5’ end of the TATA-box region was made to assess its unique activity in affecting transgene expression. Our results showed that TFBSs are essential for effective transgene expression in mouse liver after a hydrodynamic tail vein gene delivery. Plasmid with a TATA-box with all TFBS deleted resulted in minimal level of gene product, 700-fold lower than the original plasmids. Reporter gene expression appears transient reaching the background level in less than 2 weeks. The more TFBS the plasmid has, the higher level of gene product was seen. Plasmid containing a single TFBS either NFκB, ATF1, CEBPA, HNF4α, or HNF1 exhibits persistent transgene expression for more than 2 months, at which animals injected with plasmids containing NFkB, ATF1, CEBPA, HNF4α, or HNF1 exhibited gene product level 60, 130, 1020, 2250, and 2076 folds higher than that of animals injected with plasmids without TFBS, respectively. Mechanism study employing real time PCR, Western Blotting, and ChIP reveals involvement of histone acetylation and the binding of acetylated histones to plasmid DNA sequences in TFBS-mediated enhancement in persistency of reporter gene expression. TFBSs for HNF4α appear most potent in recruiting H4AC and TBP proteins to the plasmids. Our results suggest that TFBSs for NFkB, ATF1, CEBPA, HNF4α, and HNF1 are important elements to include into the promoter for achieving longer-term gene expression. In addition, histone acetylation on plasmid is a probable pathway that TFBS regulate the duration of transgene expression.

101. Generation of Improved Recombinant AAV3 Serotype Vectors Using AAV3 ITRS and AAV3 REP Proteins

Although recombinant AAV3 serotype vectors were largely ignored previously, owing to their poor transduction efficiency in all cells and tissues examined, our initial observation of their selective tropism for human liver cancer cell lines and primary human hepatocytes (Mol Genet Metabol., 98: 289-299, 2009; Hum Gene Ther., 21: 1741-1747, 2010; Gene Ther., 19: 375-84, 2012), has led to renewed interest in this serotype, since AAV3 vectors and their variants have now proven to be extremely efficient in targeting human and non-human primate hepatocytes in vitro as well as in vivo (Mol Ther., 22: S2, 2014; Mol Ther., 22: S91, 2014; Nature, 506: 382-386, 2014; Hum Gene Ther., 25: 1023-1034, 2014). Previously, it wasreported that the combination of ITR8 with AAV8 capsids (AAV8/8) resulted in vectors that led to at least 2-fold increase in transgene expression in mouse liver, compared with AAV8 capsids pseudotyped with ITR2 (AAV2/8) vectors (Mol. Ther. 11: S156, 2005), and that the ITRs from AAV serotypes 1-6 were interchangeable when they are packaged into AAV8 capsids (AAV1/8 – AAV6/8), but played no role in transgene expression in murine hepatocytes in vivo (J. Virol., 80: 426-439, 2006). In our present studies, we wished to evaluate the relative contribution of the cis-acting ITR from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the recombinant AAV3 vector production and transduction. To this end, we utilized two helper plasmid: pAAVr2c3, which carries rep2 and cap3 genes and pAAVr3c3, which carries rep3 and cap3 genes. Plasmid transfection assays revealed that both AAV2 and AAV3 Rep proteins were expressed at similar levels in the presence of adenoviral helper genes. We also generated two sets of single-stranded AAV vector constructs carrying an expression cassette containing the EGFP reporter gene flanked by either ITR2 or ITR3. Plasmid transfections of the ITR2- or the ITR3-containing expression cassettes into cultured cells also revealed no difference in the extent of transgene expression. The combination of ITR3 and Rep3 resulted in ~5-fold higher rAAV3 vector titers, as determined by quantitative PCR assays. Next, purified viral stocks of AAV3 vectors were generated and designated as Rep2-ITR2 and Rep3-ITR3, respectively. Interestingly, the transduction efficiency of Rep3-ITR3 AAV3 vectors was ~4-fold higher than that of Rep2-ITR2 AAV3 vectors in a human hepatocellular carcinoma cell line, Huh7, under identical conditions. Additional studies are currently underway to evaluate the efficacy of the Rep2-ITR2 and Rep3-ITR3 vectors in a murine xenograft model in vivo. In summary, our studies document that the combined use of the AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids lead to the production of recombinant AAV3 vectors with higher titers and with higher transduction efficiency, and suggest that the use of homologous ITRs, Rep proteins, and capsids are similarly likely to be applicable to other AAV serotypes vectors for their optimal use in human gene therapy.

Anatomy of plasmid DNAs with anti-silencing elements

The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected.

Correlation between transgen expression and plasmid DNA loss in mouse liver

Transgene expression from plasmid DNA is dependent on the expression efficiency per plasmid and the amount of intranuclear plasmid. In the present study, intranuclear dispositions of two types of plasmid DNAs (i.e. the pCpGfree and pLIVE plasmids) that maintain transgene expression in mouse liver were analyzed. In addition, the relationship between transgene expression and plasmid stability in the nucleus was examined. First, the pCpGfree and pLIVE plasmid DNAs, bearing the mouse secreted alkaline phosphatase (Seap) gene, were administered into mouse liver by the hydrodynamics-based method. Next, various Seap-plasmid DNAs containing different promoters, upstream and downstream sequences, and backbones were injected into mice, and both SEAP expression and plasmid DNA amounts were monitored for 28 days. At the 14- and 28-day time points, the amount of the pCpGfree plasmid DNA was one order of magnitude less than that of the pLIVE plasmid. Meanwhile, the expression efficiency per plasmid was one order of magnitude more efficient for the pCpGfree plasmid DNA. Moreover, the administration of various Seap-plasmid DNAs revealed that negative correlations exist between plasmid stability and SEAP expression level. The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA.

Inflammation Promotes the Loss of Adeno-Associated Virus–Mediated Transgene Expression in Mouse Liver

Non-self transgenes delivered to mouse liver via adeno-associated virus (AAV) are expressed stably due to the induction of immune tolerance. However, such transgene expression has been reported to be lost in higher-order primates. We investigated whether inflammatory processes, which likely differ between species, impact the stability of transgene expression. We developed a mouse model that mimics a scenario in which a subject that has received hepatic AAV-mediated gene transfer develops subsequent, vector-unrelated, systemic inflammation. Inflammation eliminated previously stable expression of transgenes delivered by AAV; the limited tissue destruction and persistence of AAV genomes implicated pathways besides the cytotoxic T-cell response. Tumor necrosis factor-a down-regulated expression of the transgene from the AAV, indicating a role for similar inflammatory cytokines in such loss of transgene expression. Inflammation can block AAV-mediated expression of transgenes in mouse liver. The presence of inflammation might therefore affect hepatic expression of transgenes from viral vectors in humans.

Positive Correlation Between the Generation of Reactive Oxygen Species and Activation/Reactivation of Transgene Expression After Hydrodynamic Injections into Mice

Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections.

PiggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.

Reactivation of Silenced Transgene Expression in Mouse Liver by Rapid, Large-Volume Injection of Isotonic Solution

Rapid, large-volume injection, or so-called hydrodynamic injection, of naked plasmid DNA gives high transgene expression in mouse liver, and this method has been applied to liver-directed gene transfer in humans with slight modifications. To prove that injection-induced biological changes are involved in hydrodynamic injection-induced, high-level transgene expression in mouse liver, isotonic solutions were injected into mice that had received a hydrodynamic injection of plasmid DNA. Transgene expression in the liver was increased by such injections irrespective of the cDNA, promoter, and type of solution. This reactivation was repeatable and detectable even 3 months after gene transfer. Parameters required for reactivation were similar to those required for the hydrodynamic injection of plasmid DNA. Plasmid DNA-polyethyleneimine complex-based transgene expression in mouse liver was also reactivated by the same treatment. DNA microarray and quantitative RT-PCR analyses revealed that the expression of immediate-early response genes c-fos and c-jun was increased 70 and 100-fold, respectively. Activator protein (AP)-1- or nuclear factor (NF)-kappa B-dependent transgene expression was increased by an injection of isotonic solutions. These findings indicate for the first time that a rapid, large-volume injection of isotonic solution activates the transcription factors AP-1 and NF-kappa B in the liver, which in turn increases the transcription of genes delivered by hydrodynamic injection or other methods.

Reactivation of Silenced Transgene Expression in Mouse Liver by Rapid, Large-volume Injection of Isotonic Solution

Reactivation of Silenced Transgene Expression in Mouse Liver by Rapid, Large-volume Injection of Isotonic Solution

997. Immune-Mediated Loss of Transgene Expression in Mouse Liver Following Transposon Delivery

An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice.The purpose of this study was to analyze contribution of cell- mediated immune response to the clearance of liver cells expressing foreign transgenes. We used high-pressure (hydrodynamic), tail-vein injection to deliver the transposon vectors pT2/mCAGGS-(hIDUA/hGUSB/Luc)//Ub-SB11 to the liver of WT C57Bl/6 mice. The efficiency of injection was evaluated 24 hr postinjection for transgene expression by either enzyme assay for IDUA or GUSB in plasma or in vivo imaging of luciferase (Luc). Mice were euthanized at 7, 10, 14, 17, 21 and 28 days and livers were extracted. Adjacent 5-μm sections from each liver were analyzed for transgene expression (histochemical staining for GUSB), for the presence of cellular infiltrates (H&E staining), and for the presence of CD4+ and CD8+ lymphocytes (immunohistochemical staining). In addition, the maintenance of the hIDUA, hGUSB and Luc transgene, as well as persistence of the transposon plasmid, were determined by PCR. Our results show the temporal relationship between expression of the therapeutic transgene and induction of immune response in the livers of transgene-treated mice.P.B. Hackett and R.S. McIvor have financial interest in Discovery Genomics, Inc. An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice. The purpose of this study was to analyze contribution of cell- mediated immune response to the clearance of liver cells expressing foreign transgenes. We used high-pressure (hydrodynamic), tail-vein injection to deliver the transposon vectors pT2/mCAGGS-(hIDUA/hGUSB/Luc)//Ub-SB11 to the liver of WT C57Bl/6 mice. The efficiency of injection was evaluated 24 hr postinjection for transgene expression by either enzyme assay for IDUA or GUSB in plasma or in vivo imaging of luciferase (Luc). Mice were euthanized at 7, 10, 14, 17, 21 and 28 days and livers were extracted. Adjacent 5-μm sections from each liver were analyzed for transgene expression (histochemical staining for GUSB), for the presence of cellular infiltrates (H&E staining), and for the presence of CD4+ and CD8+ lymphocytes (immunohistochemical staining). In addition, the maintenance of the hIDUA, hGUSB and Luc transgene, as well as persistence of the transposon plasmid, were determined by PCR. Our results show the temporal relationship between expression of the therapeutic transgene and induction of immune response in the livers of transgene-treated mice. P.B. Hackett and R.S. McIvor have financial interest in Discovery Genomics, Inc.

Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), α-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken β actin (ACT), nuclear factor κ B (NFκB), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within the first 5–7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein–Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.

1142. Regulation of Transgene Expression in Mouse Liver

1142. Regulation of Transgene Expression in Mouse Liver

Linear DNAs Concatemerize in Vivo and Result in Sustained Transgene Expression in Mouse Liver

The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human α1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 μg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

Adenoviral vector-mediated gene therapy in the mouse lung: no role of Fas-Fas ligand interactions for elimination of transgene expression in bronchioepithelial cells.

The early successes of adenoviral vector-mediated gene therapies in the lung have been hampered by the immune response directed against viral proteins and transgene product. Intratracheal administration of adenovirus vector in immune-competent mice transduces bronchioepithelial cells of lung extremely efficiently; however, transgene expression is eliminated within 2 weeks. Extinction of transgene expression has been attributed to infiltrating cytotoxic T lymphocytes (CTLs). Fas-Fas ligand (Fas-FasL) interactions play a critical role in the effector function of the CTL killing. We have previously demonstrated that this interacting pair of molecules plays a critical role in elimination of transgene expression in mouse liver. In this study we investigated the role of Fas-FasL interactions in CTL effector functions in vivo in mouse lung. Analyses of these molecules in lung showed constitutive expression of Fas in bronchiooepithelial cells. On the other hand, FasL was inducible in the bronchioepithelial cells after adenovirus vector treatment. The in vivo role of the Fas-FasL interactions was determined by adoptive transfer of splenocytes from normal immune-competent mice to Fas-deficient mice (B6-lpr); likewise, the function of FasL on CTLs was analyzed by the ability of splenocytes from FasL-deficient mice (B6-gld) to eliminate transgene expression in Rag1-deficient mice. These studies demonstrate that despite expression of Fas and FasL in bronchioepithelial cells and CTLs, these interacting molecules do not play a critical role in elimination of transgene expression in the lung.

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration.

We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LC8cCARP, for use in the Cre/loxP system (Parks et al. Proc Natl Acad Sci USA, 1996; 93: 13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our original helper virus (based on serotype 5, Ad5LC8cluc) showed that the two helper viruses amplified hdAd with a similar efficiency, and resulted in a similar yield and purity after large-scale preparation of vector. In vitro, the resulting hdAd2 had a similar transduction efficiency and expression kinetics of transgene (beta-gal) as the hdAd5. An important feature of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently, vectors produced with help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, and vectors produced with Ad5 helper were resistant to neutralizing antibodies against Ad2. Analysis of transgene expression in mouse liver after intravenous injection of the Ad2-based hdAd showed that the vector could efficiently transduce the liver, and produce high levels of a foreign transgene, similar to those expressed by the hdAd generated with the Ad5 helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did not cross-react with hdAd5. To determine if successful repeat Ad vector administration could be achieved by sequential use of alternative Ad serotypes, we injected mice with hdAd2 (hSEAP) followed 3 months later by a lacZ-expressing hdAd of either the same or different serotype. Repeated administration of hdAd2 resulted in a 30- to 100-fold reduction in transgene expression compared with naive animals. In contrast, no decrease in transgene expression was observed when the second vector was of a different serotype. These results demonstrate that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.