PubMed Health⌕ Search

Biomedical subjects

Charles Coutelle

Publications and source records attributed to Charles Coutelle.

At least 19 recordsLinked to original sources
PubMed2006

Covalent attachment of multifunctional chimeric terminal proteins to 5' DNA ends: A potential new strategy for assembly of synthetic therapeutic gene vectors.

The generation of synthetic therapeutic gene vectors requires the coupling of DNA to transfer-promoting peptides including cellular receptor ligands, protein transduction domains, hydrophobic peptides for attachment to lipid membranes, nuclear localisation signals, cytoskeleton attachment motifs, nuclear matrix association elements and immune evasion moieties. Existing methods of peptide-DNA joining often interfere with transgene expression and, therefore, are inadequate for production of effective therapeutic vector complexes, particularly destined for gene delivery in the challenging environment in vivo. However, there is a natural mechanism for rigid coupling of polypeptides with DNA. Some bacterial and eukaryotic linear plasmids, adenoviruses and a number of bacteriophages including phi29 of Bacillus subtilis and PRD1 of Escherichia coli use terminal proteins covalently bound to 5' DNA ends to prime replication. Inverted terminal DNA repeats, normally short DNA sequences, contain all the sequences required in cis for the covalent coupling reaction. The complex of the terminal protein, DNA polymerase and some known auxiliary proteins supplies sufficient trans-functions, thus enabling simple linking of the terminal proteins to DNA in vitro. We hypothesise that chimeric fusion proteins, constructed on the basis of terminal proteins of adenoviruses, linear plasmids or bacteriophages with protein-primed replication, can on the one hand retain the ability to bind covalently 5' DNA termini in conditions established previously for protein-primed replication in vitro, and on the other hand confer gene transfer facilitating properties and enhanced longevity of efficient transgene expression. Terminal localisation of the chimeric proteins can ensure that they do not interfere with transgene transcription. At the same time a covalent bond between polypeptide and DNA can provide rigid coupling ensuring their stable association en route to nuclei. Bound to 5'-ends of the delivered DNA, terminal protein-based chimeras could also protect the vector DNA from 5'-3' and possibly 3'-5' exonuclease attack, thus limiting its intracellular degradation and increasing longevity of transgene expression. Our hypothesis can be tested by measuring the gene transfer efficiency of the novel complexes containing linear DNA fragments with covalently linked multifunctional chimeric terminal proteins, using previously described synthetic gene vectors as standards.

5' Untranslated Regions↗
PubMed2006

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.

Animals↗
PubMed2006

RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA.

BACKGROUND: Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. RESULTS: We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination. CONCLUSION: Using transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.

Chromosomes, Bacterial↗
PubMed2006

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo.

BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.

Animals↗
PubMed2005

Coupled analysis of bacterial transformants and ligation mixture by duplex PCR enables detection of fatal instability of a nascent recombinant plasmid.

When a DNA cloning experiment fails, it is often difficult to distinguish between an inadequate cloning protocol and instability of the new recombinant plasmid. The identification of plasmid instability is particularly challenging when the instability is fatal and no DNA of the expected construct can be isolated. We have effectively addressed this problem by employment of duplex PCR (insert-insert, vector-insert) to analyse both the ligation mixture and the resultant bacterial transformants. Using this approach we found a fatal maintenance instability of one of the plasmids generated during subcloning of the cDNA for human LDLR in Escherichia coli STBL2. The described duplex PCR screening method allows monitoring of the fate of nascent recombinant plasmid from ligation, through the initial bacterial colony and the subsequent overnight culture.

Cloning, Molecular↗
PubMed2005

Synthesis and application of integrin targeting lipopeptides in targeted gene delivery.

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl-L-alpha-phosphatidylethanolamine), mu peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target alpha(9)beta(1)-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. "Targeted" LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.

Animals↗
PubMed2005

Complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice.

BACKGROUND: The baculovirus Autographa californica nucleo-polyhedrosis virus (AcNPV) is an alternative to other viral vectors for hepatic gene delivery. A barrier to AcNPV being used in vivo is its susceptibility to inactivation by serum complement 1. In vivo utility has only been demonstrated using methods that avoid contact with serum 2-5. We have studied the complement pathways involved in baculovirus inactivation in vitro and the systemic administration of baculovirus vectors in vivo, with the co-administration of the complement inhibitor, soluble complement inhibitor 1 (sCR1). RESULTS: EDTA increased baculovirus survival in human serum more than EGTA showing that both the alternative and classical pathways of complement are activated. Depleting serum of IgM increased survival, whereas reconstitution with pooled IgM restored activity against baculovirus, suggesting naturally occurring IgM antibodies with affinity for baculovirus may be partially responsible for complement activation. Intraportal administration of baculovirus led to hepatic expression when the complement inhibitor sCR1 (soluble complement receptor type 1) was co-administered by tail vein injection; however, liver histology showed hepatic necrosis. Without co-administration of sCR1, intraportal infusion of baculovirus was fatal within 24 h. Histology demonstrated massive hepatic necrosis. Yolk sac vein injection of baculovirus was associated with fetal death. CONCLUSIONS: Transgene expression was demonstrated following intraportal infusion of recombinant baculovirus vectors in combination with sCR1; however, our experiments suggest a significant associated toxicity.

Animals↗
PubMed2005

Targeting the respiratory muscles of fetal sheep for prenatal gene therapy for Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a lethal degenerative muscular disease. Fetal gene therapy may correct the primary genetic defect. Our aim was to achieve expression of a reporter gene in the respiratory muscles of early gestation fetal sheep. STUDY DESIGN: An adenovirus vector containing the beta-galactosidase reporter gene (AdRSVbetagal) was injected into the thoracic musculature (n = 3) and pleural cavity (n = 6) of fetal sheep (61-67 days' gestation) under ultrasound guidance. Tissues were harvested after 48 hours and site and intensity of beta-galactosidase expression were assessed. RESULTS: Limited transgene expression observed after a single injection was improved by multiple injections, but remained localized. Ultrasound-guided creation of a hydrothorax led to an increase in the intensity of beta-galactosidase expression (ELISA). X-gal staining and immunohistochemistry showed that vector spread was confined to the innermost intercostal musculature. CONCLUSION: Ultrasound-guided injection can deliver gene therapy vectors to the fetal pleural cavity and achieve transduction of the respiratory muscles.

Adenoviridae↗
PubMed2005

In utero gene therapy: current challenges and perspectives.

Over the past few years, considerable progress in prenatal diagnosis and surgery combined with improvements in vector design vindicate a reappraisal of the feasibility of in utero gene therapy for serious monogenetic diseases. As adult gene therapy gathers pace, several apparent obstacles to its application as a treatment may be overcome by pre- or early postnatal treatment. This review will examine the concepts and practice of prenatal vector administration. We aim to highlight the advantages of early therapeutic intervention focusing on diseases that could benefit greatly from a prenatal gene therapy approach. We will pay special attention to the strategies and vectors that are most likely to be used for this application and will speculate on their expected developments for the near future.

Animals↗
PubMed2005

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.

Animals↗
PubMed2004

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.

Animals↗
PubMed2003

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein.

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

Active Transport, Cell Nucleus↗
PubMed2003

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.

Adenoviridae↗
PubMed2003

Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero.

In utero gene therapy may provide treatment of genetic diseases before significant organ damage, allow permanent genetic correction by reaching stem cell populations, and provide immune tolerance against the therapeutic transgenes and vectors. We have used percutaneous ultrasound-guided injection as a minimally invasive fetal procedure. First-generation adenoviruses encoding the nuclear localizing beta-galactosidase reporter gene or the human factor IX (hFIX) gene, or colloidal carbon were delivered via the umbilical vein (UV, n = 4), heart (intracardiac [IC], n = 2), liver parenchyma (intrahepatic [HE], n = 11), peritoneal cavity (intraperitoneal [IP], n = 14), skeletal musculature ([intramuscular [IM], n = 11), or the amniotic cavity (intraamniotic [IA], n = 14) to early-gestation fetal sheep (0.3 gestation = day 33-61). Postmortem analysis was performed at 2, 9, or 28 days after injection. Although fetal survival was between 77% and 91% for IP, HE, IA, and IM routes, no fetuses survived UV or IC procedures. The hFIX levels reaching 1900 and 401 ng/ml (IP), 30 ng/ml (HE), 66.5 and 39 ng/ml (IA), and 83 and 65.5 ng/ml (IM), respectively, were determined 2 days after injection and decreased at birth to 16.5 ng/ml (IP), 7 ng/ml (HE), 4.5 ng/ml (IA), and 4 and 0 ng/ml (IM). Polymerase chain reaction (PCR) and immunohistochemistry showed broadest hFIX transgene spread and highest localised beta-galactosidase expression, respectively, after IP administration. Antibodies were observed against vector but not against hFIX.

Adenoviridae↗
PubMed2003

Enhancement of adenovirus-mediated gene transfer to the airways by DEAE dextran and sodium caprate in vivo.

Gene transfer to the trachea and airways by adenoviral vectors is limited by the basolateral localization of viral receptors, resulting in relatively low levels of transduction. Modification of paracellular permeability by sodium caprate, which opens tight junctions, enhances gene transfer from the apical side of cultured human airway epithelial cells. Based on this observation we investigated whether Na-caprate could also increase gene transfer when applied to the luminal surface of the airway epithelia in vivo and compared these results with EGTA, which has previously been shown to enhance adenovirus transduction. Transgene expression in the trachea and upper airways was increased 25-fold by a 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways EGTA had no effect, whereas expression of beta-gal was increased 3-fold by Na-caprate. When the adenovirus was complexed with DEAE dextran, transduction of the airway epithelia after Na-caprate pretreatment was increased 45-fold over virus alone. In conclusion, Na-caprate facilitates gene transfer to airway epithelia, particularly when adenovirus is complexed with DEAE dextran, and may in future be used in a clinical setting to enhance the efficiency of vectors for gene therapy of cystic fibrosis via airway delivery.

Adenoviridae↗
PubMed2003

High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides.

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.

Amino Acid Sequence↗
PubMed2002

In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor.

The fundamental hypotheses behind fetal gene therapy are that it may be possible (1) to achieve immune tolerance of transgene product and, perhaps, vector; (2) to target cells and tissues that are inaccessible in adult life; (3) to transduce a high percentage of rapidly proliferating cells, and in particular stem cells, with relatively low absolute virus doses leading to clonal transgene amplification by integrating vectors; and (4) to prevent early disease manifestation of genetic diseases. This study provides evidence vindicating the first hypothesis; namely, that intravascular prenatal administration of an adenoviral vector carrying the human factor IX (hFIX) transgene can induce immune tolerance of the transgenic protein. Following repeated hFIX protein injection into adult mice, after prenatal vector injection, we found persistence of blood hFIX and absence of hFIX antibodies in 5 of 9 mice. Furthermore, there was substantial hFIX expression after each of 2 reinjections of vector without detection of hFIX antibodies. In contrast, all adult mice that had not been treated prenatally showed a rapid loss of the injected hFIX and the development of high hFIX antibody levels, both clear manifestations of a strong immune reaction.

Adenoviridae↗