Delayed hematopoietic recovery after auto-SCT in patients receiving arsenic trioxide-based therapy for acute promyelocytic leukemia: a multi-center analysis.

A potential link between arsenic (ATO)-based therapy and delayed hematopoietic recovery after autologous hematopoietic SCT (HSCT) for acute promyelocytic leukemia (APL) has previously been reported. We retrospectively reviewed the clinical histories of 58 patients undergoing autologous HSCT for APL at 21 institutions in the United States and Japan. Thirty-three (56%) of the patients received ATO-based therapy prior to stem cell collection. Delayed neutrophil engraftment occurred in 10 patients (17%): 9 of the 10 patients (90%) received prior ATO (representing 27% of all ATO-treated patients), compared with 1 of the 10 patients (10%) not previously treated with ATO (representing 4% of all ATO-naïve patients; P<0.001). Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer times to ANC recovery (median 12 days vs 9 days, P<0.001). In multivariate analysis, the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.87; P<0.001). Of the available stem cell aliquots from APL patients, the median viable post-thaw CD34+ cell recovery was significantly lower than that of cryopreserved autologous stem cell products from patients with non-APL AML. Our findings suggest that ATO exposure prior to CD34+ cell harvest has deleterious effects on hematopoietic recovery after autologous HSCT.

Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

Role of antigen-specific regulatory CD4+CD25+ T cells in tolerance induction after neonatal IP administration of AAV-hF.IX.

Neonatal AAV8-mediated Factor IX (F.IX) gene delivery was applied as a model for exploring mechanisms of tolerance induction during immune ontogeny. Intraperitoneal delivery of AAV8/ Factor IX (hF.IX) during weeks 1-4 of life, over a 20-fold dose range, directed stable hF.IX expression, correction of coagulopathy in F.IX-null hemophilia B mice, and induction of tolerance to hF.IX; however, only primary injection at 1-2 days of life enabled increasing AAV8-mediated hF.IX expression after re-administration, due to the absence of anti-viral capsid antibodies. Adoptive splenocyte transfer from tolerized mice demonstrated induction of CD4(+)CD25(+) T regulatory (T(reg)) populations that specifically suppressed anti-hF.IX antibody responses, but not responses to third party antigen. Induction of hF.IX antibodies was only observed in tolerized mice after in vivo CD4(+)CD25(+) cell depletion and hF.IX challenge. Thus, primary injection of AAV during a critical period in the first week of life does not elicit antiviral responses, enabling re-administration of AAV and augmentation of hF.IX levels. Expansion of hF.IX-specific CD4(+)CD25(+) T(regs) has a major role in tolerance induction early in immune ontogeny. Neonatal gene transfer provides a useful approach for defining the ontogeny of immune responses and may suggest approaches for inducing tolerance in the context of genetic therapies.

In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.

Reexpression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration.

Adenovirus-mediated gene delivery is limited by the induction of immune responses that produce toxicity and prevent reexpression. To determine whether adenoviral delivery in the preimmune fetus would produce tolerance, we assessed luciferase (luc) expression following sequential pre- and postnatal adenoviral-mediated gene delivery. Day 15 fetuses were injected intrahepatically with 1 x 10(7) pfu of an adenoviral-luc vector (Ad-luc). Following in utero injection, hepatic luc expression persisted 1 month postnatally. No humoral response to adenovirus or luc was detected. Adult mice, previously injected in utero, were reinjected intravenously with 5 x 10(8) pfu of Ad-luc at 3 months of age and again at 6 months with either 5 x 10(8) pfu of Ad-luc or cationic liposome-DNA complexes (CLDC). Following the first postnatal injection, animals injected in utero had levels of luc comparable to those of age-matched naive controls. However, both control and experimental animals subsequently developed antibodies to adenovirus and luc. No further expression was achieved with a second postnatal injection of Ad-luc or with delivery of CLDC-luc. These studies demonstrate that the delivery of adenoviral vectors in utero at E15 does not elicit an immune response. However, delivery of recombinant adenovirus postnatally results in brisk and limiting immune responses regardless of the in utero exposure.

Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin.

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Fetal gene transfer by transuterine injection of cationic liposome-DNA complexes.

In utero injection of cationic liposome-DNA complexes (CLDCs) containing chloramphenicol acetyltransferase, beta-galactosidase (beta-gal), or human granulocyte colony-stimulating factor (hG-CSF) expression plasmids produced high-level gene expression in fetal rats. Tissues adjacent to the injection site exhibited the highest levels of gene expression. Chloramphenicol acetyltransferase expression persisted for at least 14 days and was reexpressed following postnatal reinjection of CLDCs. Intraperitoneal administration of the hG-CSF gene produced high serum hG-CSF levels. X-gal staining demonstrated widespread beta-gal expression in multiple fetal tissues and cell types. No toxic or inflammatory responses were observed, nor was there evidence of fetal-maternal or maternal-fetal gene transfer, suggesting that CLDCs may provide a useful alternative to viral vectors for in utero gene transfer.

Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero.

Development of in utero gene transfer approaches may provide therapies for genetic disorders with perinatal morbidity. In hemophilia A, prenatal and postnatal bleeding may be catastrophic, and modest increments in factor VIII (FVIII) activity are therapeutic. We performed transuterine i.p. gene transfer at day 15 of gestation in a murine model of hemophilia A. Normal, carrier (X(H)X), and FVIII-deficient (X(H)Y and X(H)X(H)) fetuses injected with adenoviral vectors carrying luciferase or beta-galactosidase reporter genes showed high-level gene expression with 91% fetal survival. The live-born rates of normal and FVIII-deficient animals injected in utero with adenovirus murine FVIII (3.3 x 10(5) plaque-forming units) was 87%. FVIII activity in plasma was 50.7 +/- 10.5% of normal levels at day 2 of life, 7.2 +/- 2.2% by day 15 of life, and no longer detectable at day 21 of life in hemophilic animals. Injection of higher doses of murine FVIII adenovirus at embryonic day 15 produced supranormal levels of FVIII activity in the neonatal period. PCR analysis identified viral genomes primarily in the liver, intestine, and spleen, although adenoviral DNA was detected in distal tissues when higher doses of adenovirus were administered. These studies show that transuterine i.p. injection of adenoviral vectors produces therapeutic levels of circulating FVIII throughout the neonatal period. The future development of efficient and persisting vectors that produce long-term gene expression may allow for in utero correction of genetic diseases originating in the fetal liver, hematopoietic stem cells, as well as other tissues.

Adenovirus-mediated gene transfer to the peritoneum and hepatic parenchyma of fetal mice in utero.

BACKGROUND: The development of effective gene transfer in utero will provide alternative approaches to the treatment of genetic disorders. For many disorders, the fetal liver and peritoneum are important target tissues. Our goals were to compare the tissue sites and duration of transferred gene expression after intraperitoneal (i.p.) or intrahepatic adenoviral-mediated gene transfer in utero in the developing murine fetus. METHODS: Day 15 CD-1 fetuses were injected intrahepatically or intraperitoneally with recombinant adenoviruses containing the luciferase or beta-galactosidase reporter gene. Tissue levels of luciferase were quantitated, or tissues were examined for X-gal staining. RESULTS: Luciferase expression was observed in multiple fetal tissues (including brain, intestine, liver, and lung) and persisted up to 32 days after intrahepatic delivery. Significant hepatic tropism was demonstrated. CONCLUSIONS: Intrahepatic and intraperitoneal injection in utero results in transduction of multiple tissues in the developing murine fetus. Transuterine injection of fetal mice via intrahepatic and intraperitoneal routes provides a valuable model for assessing the efficacy of gene delivery vectors in the prenatal treatment of genetic disorders. These studies demonstrate that hepatic and intraperitoneal gene transfer to the developing murine fetus is feasible and may provide therapeutic levels of proteins during fetal development.

Adenovirus-mediated gene transfer in the midgestation fetal mouse.

BACKGROUND: The development of strategies for gene transfer in utero will make possible the amelioration, and eventually the cure, of genetic diseases associated with pre- and postnatal morbidity and mortality. We have developed a murine model for in utero, intrahepatic, adenovirus-mediated gene transfer in Day 15 fetuses and compared the level and distribution of luciferase reporter gene expression in newborns with those observed in adult animals injected intravenously. MATERIAL AND METHODS: CD-1 fetuses underwent intrahepatic injection on Day 15 of gestation with 1 x 10(7) particle-forming units (PFU) of an E1- and E3-deleted recombinant adenovirus containing the luciferase reporter gene or with normal saline. At birth, pups were euthanized, and the brain, heart, intestine, liver, lungs, and spleen harvested and analyzed for luciferase activity. RESULTS: Two adenovirus-injected litters proceeded to term and one female aborted. Tissues from 10 newborn mice in the experimental group and 5 newborns in the control group were analyzed; tissues from the remaining newborns were reserved for other studies. High-level luciferase expression was detected in all adenovirus-injected newborn livers. Lower levels of luciferase activity were detected in distant organs. Hepatic toxicity as determined by serum transaminase elevations was observed in adult, but not in newborn mice previously injected with the adeno-luciferase virus. CONCLUSIONS: In utero intrahepatic gene delivery with adenoviral vectors in the developing murine fetus is feasible and produces high-level gene expression. These studies suggest that viral and nonviral gene delivery vectors may be useful in the development of future approaches to prenatal treatment of genetic disorders.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb betas-globin yeast artificial chromosome: A mouse model of sickle cell anemia.

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine alpha- and beta-globin genes and substitution with human alpha and betas transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human alpha-, gamma-, and beta-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human beta-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the gamma- and beta-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb betas yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

The human beta globin locus introduced by YAC transfer exhibits a specific and reproducible pattern of developmental regulation in transgenic mice.

The human beta globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult beta globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the beta globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from gamma to beta globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human beta globin mRNA levels between lines. Human beta globin mRNA levels were twofold to fourfold lower than that of mouse betamaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the beta globin locus.

Silencing of human fetal globin expression is impaired in the absence of the adult beta-globin gene activator protein EKLF.

Globin genes are subject to tissue-specific and developmental stage-specific regulation. A switch from human fetal (gamma)-to adult (beta)-globin expression occurs within erythroid precursor cells of the adult lineage. Previously we and others showed by targeted gene disruption that the zinc finger gene, erythroid Krüppel-like factor (EKLF), is required for expression of the beta-globin gene in mice, presumably through interaction with a high-affinity binding site in the proximal promoter. To examine the role of EKLF in the developmental regulation of the human gamma-globin gene we interbred EKLF heterozygotes (+/-) with mice harboring a human beta-globin yeast artificial chromosome transgene. We find that in the absence of EKLF, while human beta-globin expression is dramatically reduced, gamma-globin transcripts are elevated approximately 5-fold. Impaired silencing of gamma-globin expression identifies EKLF as the first transcription factor participating quantitatively in the gamma-globin to beta-globin switch. Our findings are compatible with a competitive model of switching in which EKLF mediates an adult stage-specific interaction between the beta-globin gene promoter and the locus control region that excludes the gamma-globin gene.

Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice.

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region.

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.