Abstract LBA-5
Inherited hematologic disorders have the potential to be effectively treated by gene therapy, with recent successes reported for several genetic disorders using viral vector-mediated ...gene transfer (ADA-SCID, NEJM 2009; β-thalassemia, Nature 2010). However, these trials and others illustrate some of the disadvantages and risks of using viral vector-based gene addition strategies, including loss of endogenous gene regulation and random insertion leading to potential for insertional mutagenesis. An alternative approach is gene correction, where in situ correction of a gene mutation allows endogenous gene regulation and decreases risks related to random integration. Gene correction is based on gene targeting, the therapeutic utility of which has historically been limited to mouse embryonic stem cells due to low homologous recombination rates in other cell types. However, a recently developed class of fusion proteins, zinc finger nucleases (ZFNs), have been shown to increase targeting efficiency 2–3 logs by inducing site-specific DNA double strand breaks at the intended targeting site. ZFNs have permitted high efficiency therapeutic gene targeting in a variety of cultured cells previously thought intractable to these processes, but ZFN-mediated gene correction has yet to be successfully achieved in vivo in an animal model of disease. Here we show ZFN-mediated therapeutic gene targeting of a mutated F9 gene in vivo, resulting in phenotypic correction of a mouse model of hemophilia B (HB). We first generated ZFNs targeting intron 1 of the human F9 gene (F9 ZFNs). We hypothesized the F9 ZFNs would mediate insertion of a wild-type F9 exons 2–8 minigene into intron 1 via gene targeting, thus bypassing the 95% of F9 mutations that occur in exons 2–8. We next generated a humanized HB mouse model with a deletion of the mouse F9 gene and knock-in (at the ROSA 26 locus) of a catalytic domain-deleted human F9 mini-gene (hF9mut) transgene. Adeno-associated viral (AAV) vector delivery of the F9 ZFNs to hF9mut mouse liver resulted in cleavage of the intron 1 target site in 45% of hepatocytes. We then generated an AAV donor vector containing a w.t. exons 2–8 insert flanked by arms of homology. Co-delivery of the AAV-ZFN and AAV-donor vectors to neonatal hF9mut mice (n=16) resulted in circulating F.IX levels of 120–350 ng/mL (2-7% of normal), whereas mice receiving AAV-ZFN alone (n=17) or AAV-mock & AAV-donor (n=15) had no detectable F.IX expression (detection limit 15 ng/mL), or <25 ng/mL F.IX, respectively. PCR analysis of liver DNA from ZFN+donor mice demonstrated genomic evidence of gene targeting at a rate of 2–7% of alleles. F.IX expression in ZFN+donor mice was shown to be stable after 5 months, with follow-up ongoing. In addition, there was no loss of expression following partial hepatectomy, which causes loss of expression from non-integrated episomes upon subsequent hepatocyte proliferation. F.IX expression was also shown to be specific, as opposed to resulting from random integration, as mice lacking the hF9mut gene averaged less than 30 ng/mL after receiving AAV-ZFN and AAV-donor. hF.IX RT-PCR on 10 different tissues confirmed liver-specific expression. To assess phenotypic correction, we performed aPTTs on mice that received ZFN+donor or mock+donor, as well as wild-type (WT) mice and HB mice. WT mice averaged 36 seconds, ZFN+donor mice averaged 44 seconds, mock+donor mice averaged 60 seconds, and HB mice averaged 67 seconds. There was no significant difference in aPTT between WT and ZFN+donor, or mock+donor and HB (p = 0.086 and 0.11, respectively). However, the aPTT for ZFN+donor mice was significantly shortened compared to mock+donor mice (p=0.0014), demonstrating phenotypic correction of the defect in clot formation in HB mice. To our knowledge this is the first demonstration of ZFN-driven gene correction in vivo, and the first demonstration of the in vivo use of ZFNs to correct an animal model of human disease. These results establish a novel paradigm for in vivo gene correction as a method for treating inherited hematologic diseases.
Doyon:Sangamo Biosciences: Employment. Li:Sangamo Biosciences: Employment. Wong:Sangamo Biosciences: Employment. Paschon:Sangamo Biosciences: Employment. Rebar:Sangamo Biosciences: Employment. Gregory:Sangamo Biosciences: Employment. Holmes:Sangamo: Employment. High:Sangamo Biosciences: Consultancy; Children's Hospital of Philadelphia: Patents & Royalties.
Abstract 2465
Poster Board II-442
Gene therapy for Hemophilia B is a promising alternative to recombinant protein therapy. Long-term expression of coagulation factor IX via stable gene transfer could ...reduce costs and risks associated with intravenous recombinant factor IX infusions. Liver-directed, adeno-associated virus (AAV) vector-mediated gene transfer of factor IX (FIX) is a gene therapy strategy currently used in two human clinical trials. Our group is performing one of these trials using an AAV vector (AAV-hFIX16) expressing human FIX from a liver-specific promoter. Hepatic artery delivery of AAV-hFIX16 has shown short-term efficacy and disease correction. A fundamental issue facing clinical gene transfer has to do with risk related to vector integration. Though AAV vector DNA is predominantly episomal in transduced cells, chromosomal integration can occur, and consequences of AAV genotoxicity at the molecular level in in vivo systems require further analysis. Here we present the results of a large-scale longitudinal study in which we followed 120 male, wild-type C57BL/6 mice for 18 months after portal vein injection of either 5e12 vg/kg of AAV-hFIX16 (60 mice), 1e14 vg/kg of AAV-hFIX16 (20 mice), 1e14 vc/kg of empty AAV capsid (20 mice), or saline (20 mice). At 18 months we found circulating hFIX levels of 12 ug/mL in mice receiving the lower AAV dose, and 25 ug/mL in mice receiving the higher AAV dose, with no detectable hFIX in mice receiving empty capsid or saline. To assess the risk of insertional mutagenesis in cells transduced by AAV vector, we analyzed the incidence of hepatocellular carcinoma (HCC). We found an HCC incidence rate of 9.1% in untreated mice, 0% in mice receiving saline, 0% in mice receiving empty capsid, 3.8% in mice receiving the lower AAV dose, and 12.5% in mice receiving the higher AAV dose, resulting in a p-value of 0.64 when comparing both AAV groups to controls. We quantified vector genome copy number by qPCR in the HCC's occurring in mice treated with AAV and compared them to vector genome copy number in normal tissue adjacent to the tumors and found no statistically significant difference (p=0.3). We then used LM-PCR to recover vector-chromosome integration junctions from tumor tissue and normal adjacent tissue. We were able to clone 249 unique integrants from tumor tissue and 862 unique integrants from normal adjacent tissue. This yields an average of 0.00018 unique integrants per diploid genome in tumor tissue and 0.00064 unique integrants per diploid genome in normal adjacent tissue. When determining integration profiles within the mouse genome, we found that integrants in both tumor tissue and normal adjacent tissue had a preference for integrating within genes, particularly within exons, as well as within CpG-rich regions. In addition, we found that integrants in both tumor tissue and normal adjacent tissue were more likely than not to be located within 50kb of the 5′ end of an oncogene. While this preference was stronger for integrants in normal adjacent tissue (p=6.3e-5) than integrants in tumor tissue (p=7.7e-2), it was even more likely for the HIV virus to integrate within 50kb of the 5′ end of an oncogene (p=4.1e-11). Functional consequences of these sites of AAV vector integration are not yet known, but are currently being investigated further. In conclusion, our large-scale prospective study of mice receiving AAV-hFIX16 did not demonstrate a statistically significant difference in HCC incidence between AAV-treated groups and control groups, although there was a trend of increasing HCC incidence with very high AAV doses. When analyzing tumors from mice treated with AAV, we found no relationship between vector genome copy number and tumor formation. When analyzing integrated vector in tumor tissue and normal adjacent tissue, we found that integrants in both normal adjacent tissue and tumor tissue had less likelihood for localizing within 50kb of the 5' end of oncogenes than HIV.
Wright:Genzyme: Consultancy; Tacere: Consultancy.
In this paper, we report on the formation and properties of a water-stabilized dimer comprising calix4arene-guanosine conjugate cG 2. The 1,3-alternate calixarene cG 2 was poorly soluble in dry CDCl3 ...and gave an ill-resolved NMR spectrum, consistent with its nonspecific aggregation. The compound was much more soluble in water-saturated CDCl3. Two sets of well-resolved 1H NMR signals for the guanosine residues in cG 2, present in a 1:1 ratio, indicated that the compound's D 2 symmetry had been broken and provided the first hint that cG 2 dimerizes in water-saturated CDCl3. The resulting dimer, (cG 2)2·(H2O) n , has a unique property: it extracts alkali halide salts from water into organic solution. This dimer is a rare example of a self-assembled ion pair receptor. The identity of the (cG 2)2·NaCl·(H2O) n dimer was confirmed by comparing its self-diffusion coefficient in CDCl3, determined by pulsed-field gradient NMR, with that of control compound cA 3, an adenosine analogue. The dimer's stoichiometry was also confirmed by quantitative measurement of the cation and anion using ion chromatrography. Two-dimensional NMR and ion-induced NMR shifts indicated that the cation binding site is formed by an intermolecular G-quartet and the anion binding site is provided by the 5‘-amide NH groups. Once bound, the salt increases the dimer's thermal stability. Both 1H NMR and ion chromatography measurements indicated that the cG 2 dimer has a modest selectivity for extracting K+ over Na+ and Br- over Cl-. The anion's identity also influences the association process: NaCl gives a soluble, discrete dimer whereas addition of NaBPh4 to (cG 2)2·(H2O) n leads to extensive aggregation and precipitation. This study suggests a new direction for ion pair receptors, namely, the use of molecular self-assembly. The study also highlights water's ability to stabilize a functional noncovalent assembly.
Lightweight and deployable phased arrays are important in Small-Satellite (Small-Sat) constellation systems to reduce price and increase link distance. This article proposes a Ka-band deployable ...active phased-array TX on a 4-layer liquid crystal polymer (LCP) board for a lightweight and high stowage rate solution to break through the trade-off between high antenna aperture size and small form factor in phased arrays. This article describes detailed designs of the phased array and its building blocks with the proposed 4-layer LCP substrate structure. Assembled with the beamformer ICs (BFIC), a Ka-band <inline-formula> <tex-math notation="LaTeX">8 \times 4 </tex-math></inline-formula>-element deployable active phased-array TX on the 4-layer LCP substrate is fabricated. The proposed deployable phased-array TX can steer the beam from −50° to 50°. The phased-array TX can drive 32-APSK DVB-S2 signal with a −28.7 dB EVM as well, revealing that the deployable phased-array TX performs with high phased-array performance. Furthermore, the TX achieved the lightest and thinnest phased array with 0.96 kg/m 2 areal mass and 0.9 mm thickness. This groundbreaking research sets the stage for the realization of affordable and user-centric satellite communication (SATCOM) in low-earth orbit (LEO).
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