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Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients., Graphical abstract, Combining codon and vector optimization significantly improved the efficacy of an AAV vector to treat a mouse model of homozygous familial hypercholesterolemia (HoFH). This second-generation clinical candidate vector has the potential to achieve therapeutic effects in HoFH patients and showcases translatable engineering strategies to enhance gene therapy vector efficacy.

Introductory ParagraphEfforts to improve liver gene therapy have focused on next-generation adeno-associated virus (AAV) vector capsids, transgene delivery, and immunomodulating drugs, such as corticosteroids, to avoid destructive T-cell responses. We conducted a detailed characterization of AAV transduction in nonhuman primate liver across multiple capsids and transgenes to better define interactions that may limit stable and efficient transgene expression. We show that the initial transduction of hepatocytes is high, but the transduction rapidly diminishes to a lower stable baseline of

Gene disruption via programmable, sequence-specific nucleases represents a promising gene therapy strategy in which the reduction of specific protein levels provides a therapeutic benefit. Proprotein convertase subtilisin/kexin type 9 (PCSK9), an antagonist of the low-density lipoprotein (LDL) receptor, is a suitable target for nuclease-mediated gene disruption as an approach to treat hypercholesterolemia. We sought to determine the long-term durability and safety of PCSK9 knockdown in non-human primate (NHP) liver by adeno-associated virus (AAV)-delivered meganuclease following our initial report on the feasibility of this strategy. Six previously treated NHPs and additional NHPs administered AAV-meganuclease in combination with corticosteroid treatment or an alternative AAV serotype were monitored for a period of up to 3 years. The treated NHPs exhibited a sustained reduction in circulating PCSK9 and LDL cholesterol (LDL-c) through the course of the study concomitant with stable gene editing of the PCSK9 locus. Low-frequency off-target editing remained stable, and no obvious adverse changes in histopathology of the liver were detected. We demonstrate similar on-target nuclease activity in primary human hepatocytes using a chimeric liver-humanized mouse model. These studies demonstrate that targeted in vivo gene disruption exerts a lasting therapeutic effect and provide pivotal data for safety considerations, which support clinical translation.

AAV- and CRISPR-Cas9–mediated gene targeting in neonatal mice led to clinical benefits in a mouse model of OTC deficiency., Ornithine transcarbamylase (OTC) deficiency is an X-linked urea cycle disorder associated with high mortality. Although a promising treatment for late-onset OTC deficiency, adeno-associated virus (AAV) neonatal gene therapy would only provide short-term therapeutic effects as the non-integrated genome gets lost during hepatocyte proliferation. CRISPR-Cas9-mediated homology-directed repair can correct a G-to-A mutation in 10% of OTC alleles in the livers of newborn OTC spfash mice. However, an editing vector able to correct one mutation would not be applicable for patients carrying different OTC mutations, plus expression would not be fast enough to treat a hyperammonemia crisis. Here, we describe a dual-AAV vector system that accomplishes rapid short-term expression from a non-integrated minigene and long-term expression from the site-specific integration of this minigene without any selective growth advantage for OTC-positive cells in newborns. This CRISPR-Cas9 gene-targeting approach may be applicable to all patients with OTC deficiency, irrespective of mutation and/or clinical state.

Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.

Vectors based on the clade E family member adeno-associated virus (AAV) serotype 8 have shown promise in patients with hemophilia B and have emerged as best in class for human liver gene therapies. We conducted a thorough evaluation of liver-directed gene therapy using vectors based on several natural and engineered capsids including the clade E AAVrh10 and the largely uncharacterized and phylogenically distinct AAV3B. Included in this study was a putatively superior hepatotropic capsid, AAVLK03, which is very similar to AAV3B. Vectors based on these capsids were benchmarked against AAV8 and AAV2 in a number of in vitro and in vivo model systems including C57BL/6 mice, immune-deficient mice that are partially repopulated with human hepatocytes, and nonhuman primates. Our studies in nonhuman primates and human hepatocytes demonstrated high level transduction of the clade E-derived vectors and equally high transduction with vectors based on AAV3B. In contrast to previous reports, AAVLK03 vectors are not superior to either AAV3B or AAV8. Vectors based on AAV3B should be considered for liver-directed gene therapy when administered following, or before, treatment with the serologically distinct clade E vectors.

Genome editing in non-human primates with a PCSK9-targeted meganuclease lowers cholesterol levels. Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder of the urea cycle. Hemizygous males and heterozygous females may experience life-threatening elevations of ammonia in blood and brain, leading to irreversible cognitive impairment, coma, and death. Recent evidence of acute liver failure and fibrosis/cirrhosis is also emerging in OTC-deficient patients. Here, we investigated the long-term consequences of abnormal ureagenesis in female mice heterozygous (Het) for a null mutation in the OTC gene. Two-month-old Het OTC knockout (KO) mice received a single dose of self-complementary adeno-associated virus (AAV) encoding a codon-optimized human OTC gene at 1 × 1010, 3 × 1010, or 1 × 1011 vector genome copies per mouse. We compared liver pathology from 18-month-old treated Het OTC-KO mice, age-matched untreated Het OTC-KO mice, and WT littermates, and assessed urinary orotic acid levels and vector genome copies in liver at 4, 10, and 16 months following vector administration. Het OTC-KO female mice showed evidence of liver inflammation and the eventual development of significant fibrosis. Treatment with AAV gene therapy not only corrected the underlying metabolic abnormalities, but also prevented the development of liver fibrosis. Our study demonstrates that early treatment of OTC deficiency with gene therapy may prevent clinically relevant consequences of chronic liver damage from developing.

Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence. The AAV8 capsid was chosen for all experiments due to its strong liver tropism. While EGFP is already a codon-optimized version of the original gene, both wild-type (WT) and codon-optimized (co) versions of the hOTC transgene were compared in this study. In addition, the study evaluated which of the two hOTC modifications—codon optimization or self-complementarity—would confer the highest increase in expression levels at a given dose. Interestingly, based on morphometric image analysis, it was observed that the difference in detectable expression levels between self-complementary (sc) and single-stranded (ss) hOTCco vectors was dose dependent, with a sevenfold increase in OTC-positive area using sc vectors at a dose of 3 × 10(9) genome copies (GC) per mouse, but no significant difference at a dose of 1 × 10(10) GC/mouse. In contrast, with EGFP as a transgene, the increases in expression levels when using the sc vector were observed at both the 3 × 10(9) GC/mouse and 1 × 10(10) GC/mouse doses. Furthermore, codon optimization of the hOTC transgene generated a more significant improvement in expression than the use of self-complementarity did. Overall, the results demonstrate that increases in expression levels gained by using sc vectors instead of ss vectors can vary between different transgenes, and that codon optimization of the transgene can have an even more powerful effect on the resulting expression levels.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked disorder of the urea cycle. Affected males are at risk of life threatening elevation of ammonia that can lead to irreversible cognitive impairment, coma and death. Female carriers show variable clinical manifestations depending on the level of X chromosome inactivation skewing. Histological analyses on liver samples from patients with urea cycle disorders found diffuse microvesicular steatosis, nuclear glycogen, and variable portal-to-portal bridging fibrosis. The OTC knockout (KO) model generated in our laboratory through the deletion of exons 2-3 closely mimics the severe neonatal onset form of OTCD in humans. Neonatal male OTC KO pups have elevated plasma ammonia levels due to the absence of OTC expression in the liver, and they inevitably die within 24 hours after birth. Heterozygous females breed normally, have normal plasma ammonia levels, reduced liver OTC enzyme activity, elevated urine orotic acid levels, and in some cases lower body weight compared to wild type (WT) littermates. Sirius red staining on liver samples from heterozygous mice of different ages (6, 12, and 18 months old) showed liver fibrosis in aged (18-month old) OTC-KO heterozygous female mice, similar to a liver sample from a 11-year-old OTCD patient. We then tested if gene therapy could prevent the liver fibrosis. Two-month old OTC-KO heterozygous received a single tail vein injection of a self-complementary AAV8 vector encoding a codon-optimized human OTC gene (hOTCco) at 1×10^10, 3×10^10, and 1×10^11 vector genome copies per mouse. One week following vector treatment, mice in all three vector dose groups had normal urine orotic acid levels which were maintained throughout the study (16 months). Liver samples were harvested from 18 month old treated mice for pathology analysis and compared to age-matched untreated heterozygous mice and WT littermates. All treated mice showed normal liver histology similar to WT, in contrast to the untreated heterozygous animals which had fibrosis throughout the liver. In conclusion, a single injection of AAV8sc-hOTCco vector can prevent liver fibrosis in OTC-KO heterozygous and has great potential for correction of liver fibrosis in OTCD patients.

Functional studies of embryonic epsilon-globin indicate that individuals with beta thalassemia or sickle cell disease are likely to benefit from therapeutic, transcriptional derepression of its encoding gene. The success of epsilon-globin gene-reactivation strategies, however, will be tempered by the stability that epsilon-globin mRNA exhibits in developmental stage-discordant definitive erythroid progenitors. Using cell culture and transgenic mouse model systems, we demonstrate that epsilon-globin mRNA is modestly unstable in immature, transcriptionally active erythroid cells, but that this characteristic has relatively little impact on the accumulation of epsilon-globin mRNA at subsequent stages of terminal differentiation. Importantly, the constitutive stability of epsilon-globin mRNA increases in transgenic mouse models of beta thalassemia, suggesting that epsilon- and beta-globin mRNAs are coregulated through a shared posttranscriptional mechanism. As anticipated, relevant cis-acting determinants of epsilon-globin mRNA stability map to its 3' UTR, consistent with the positioning of functionally related elements in other globin mRNAs. These studies demonstrate that posttranscriptional processes do not pose a significant practical barrier to epsilon-globin gene reactivation and, moreover, indicate that related therapeutic strategies may be particularly effective in individuals carrying beta-thalassemic gene defects.

Hemoglobin ζ 2 β 2 S is generated by substituting embryonic ζ-globin subunits for the normal α-globin components of Hb S ( α 2 β 2 S ) . This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O2-transport properties of Hb ζ 2 β 2 S have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb ζ 2 β 2 S exhibits an O2 affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O2 transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O2 affinity of intact erythrocytes from mice that express Hb ζ 2 β 2 S . Surprisingly, though, co-expression of Hb ζ 2 β 2 S leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb ζ 2 β 2 S outweigh its O2-transport liabilities. The potential structural bases for the antisickling properties of Hb ζ 2 β 2 S are discussed in the context of these new observations.

Many genetic liver diseases, including OTC deficiency, present in newborns with repeated, often lethal, metabolic crises. Adeno-associated virus (AAV) neonatal gene therapy in this setting would require multiple vector administrations to maintain the therapeutic effects because the non-integrating genome is lost as developing hepatocytes proliferate. As such, we reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9, a powerful genome-editing tool consisting of the Cas9 nuclease and a single-guide RNA (sgRNA). We developed a strategy using an AAV vector with high liver tropism (AAV8) to correct the point mutation in newborn spfash mice using Cas9 enzyme from Staphylococcus aureus (SaCas9). An animal model of OTC deficiency, the male sparse fur ash (spfash) mouse, has a G-to-A point mutation at the donor splice site at the end of exon 4 of the OTC gene, which leads to abnormal splicing and a 20-fold reduction in OTC mRNA and protein.We developed a two-vector approach to incorporate all 3 components of CRISPR/Cas9 into AAV. Vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, while vector 2 contains both the sgRNA1 sequence expressed from a U6 promoter and the 1.8 kb donor OTC DNA sequence. Spfash pups were injected intravenously on postnatal day 2 with mixtures of vector 1 and vector 2 and subsequently evaluated for indel (insertion and deletion) formation and functional correction of the spfash mutation. Following gene correction (3 and 8 weeks), indels were detected by deep sequencing in 31% of OTC alleles, and HDR-based correction of the G-to-A mutation was observed in 10% of OTC alleles. Liver sections were analyzed by immunohistochemistry for OTC expression, showing 15% OTC-positive cells at 3 weeks and 13% at 8 weeks. Direct measurements of OTC enzyme activity from liver homogenates and OTC mRNA from total cellular RNA from liver revealed similarly high levels of correction in treated animals. We further assessed the impact of gene correction on the clinical manifestations of OTC deficiency by evaluating the tolerance of spfash mice to a one-week course of high-protein diet. Spfash mice treated with the gene-editing vectors had significantly lower plasma ammonia levels and showed a survival improvement as compared with untreated spfash mice. This study provides convincing evidence for efficacy in an authentic animal model of a lethal human metabolic disease following in vivo genome editing.

Background The normal accumulation of adult α and β globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors. Methods The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3′UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure. Results We specify a tetranucleotide 3′UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult β-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development. Conclusions These data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.

Embryonic ζ- and ε-globin subunits assemble with each other and with adult α- and β-globin subunits into hemoglobin heterotetramers in both primitive and definitive erythrocytes. The properties of these hemoglobins—Hbs Gower-1 (ζ2ε2), Gower-2 (α2ε2), and Portland-2 (ζ2β2)—have been incompletely described as they are difficult to obtain in quantity from either primary human tissue or conventional expression systems. The generation of complex transgenic–knockout mice that express these hemoglobins at levels between 24% and 70% is described, as are efficient methods for their purification from mouse hemolysates. Key physiological characteristics—including P50, Hill coefficient, Bohr effect, and affinity for 2,3-BPG—were established for each of the 3 human hemoglobins. The stability of each hemoglobin in the face of mechanical, thermal, and chemical stresses was also determined. Analyses indicate that the ζ-for-α exchange distinguishing Hb Portland-2 and Hb A alters hemoglobin O2-transport capacity by increasing its P50 and decreasing its Bohr effect. By comparison, the ε-for-β exchange distinguishing Hb Gower-2 and Hb A has little impact on these same functional parameters. Hb Gower-1, assembled entirely from embryonic subunits, displays an elevated P50 level, a reduced Bohr effect, and increased 2,3-BPG binding compared to Hb A. The data support the hypothesis that Hb Gower-2, assembled from reactivated ε globin in individuals with defined hemoglobinopathies and thalassemias, would serve as a physiologically acceptable substitute for deficient or dysfunctional Hb A. In addition, the unexpected properties of Hb Gower-1 call into question a common hypothesis for its primary role in embryonic development.

Embryonic- and adult-stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (alpha2beta2) by the substitution of embryonic zeta for adult alpha globin (Hb zeta2beta2), or embryonic epsilon for adult beta globin (Hb alpha2epsilon2). Several key physiological properties of these 'semiembryonic' haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2-binding characteristics of hybrid human/mouse haemoglobins change when human alpha- or beta-globin subunits are replaced by human embryonic zeta- or epsilon-globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse alpha- or beta-globin genes, resulting in the high-level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human zeta-globin for human alpha-globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2, 3-bisphosphoglycerate (2,3-BPG), and reduced the pH-dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human epsilon-globin or human beta-globin exhibited nearly identical O2-binding properties, both in situ and in vitro, regardless of 2,3-BPG levels or ambient pH. Neither the zeta-for-alpha nor the epsilon-for-beta substitutions substantially altered binding affinity for 2,3-BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.

A variant Hb ζ2β2sthat is formed from sickle hemoglobin (Hb S; α2β2s) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2β2sinhibits the polymerization of deoxygenated Hb Sin vitroand reverses characteristic features of SCDin vivoin mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2β2), Hb ζ2β2sexhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 Å resolution crystal structure of human Hb ζ2β2sthat was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2β2sdisplays a central water cavity, a ζ1–βs2 (or ζ2–βs1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal βHis146 salt-bridge interactions, and a β-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1βs1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2β2sand, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.

Despite the availability of safe and effective recombinant FIX therapeutics, patients with Hemophilia B could obtain additional clinical benefit by having longer-acting therapeutic options. Although longer-acting recombinant protein FIX therapies are becoming available, the ultimate long-acting FIX therapy could be provided by a gene therapy approach, for example with rAAV vectors encoding FIX. Promising clinical data has been generated over the last few years in an academic setting, but these results must be replicated and extended in a commercial setting for these new therapies to become broadly available to patients.Dose-dependent inflammatory responses that impact the transgene expression have been reported in clinical trials with rAAV FIX vectors, highlighting the need for clinical rAAV vectors to have the highest possible potency such that the overall vector dose can be reduced. We have developed a potent AAV serotype rh10 vector containing a wild-type FIX gene under the control of a highly active liver specific promoter with the goal of achieving approximately 10% of normal FIX levels in patients.In preparation for a clinical trial, we tested this rAAVrh10FIX vector for aspects of efficacy and safety in the factor IX knockout (FIX-KO) mouse model of Hemophilia B. A dose-dependent increase in FIX protein and activity was observed following intravenous administration at vector doses between 1.6×1010 and 5.0×1013 GC/kg. FIX-KO mice that received AAVrh10-hFIXco at 1.6 × 1010 GC/kg achieved between 5% and 8% of normal hFIX levels at 2 weeks post-dose and maintained this level for the 90-day study period. FIX-KO mice that received rAAVrh10FIX vector at 5.0 × 1010 GC/kg achieved between 30% and 42% of normal hFIX levels at 14 days post-dose and maintained this level for the 90-day study period. From these results we can estimate that the vector dose required to achieve the stated goal of 10% of normal FIX levels of 10% would be between 1.6 × 1010 GC/kg and 5.0 × 1010 GC/kg. Expression of hFIX antigen correlated well with measured activity of hFIX antigen, with doses between 1.6 × 1010 GC/kg and 5.0 × 1010 GC/kg providing between 8% and 35% activity of normal hFIX levels.FIX-KO mice provide an excellent model to examine the effects of AAVrh10-hFIXco on hemophilia B-related complications. FIX KO mice are prone to spontaneous bleeds and often suffer from fatal hemorrhages after handling or trauma. In this study, 4 out of 7 animals in the Day 90 control group succumbed to hemophilia-related complications. Single dose intravenous administration of AAVrh10-hFIXco at doses of 1.6 × 1010, 5.0 × 1010, 1.6 × 1011, 5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 GC/kg to FIX-KO mice did not result in test article-related mortality, clinical pathology, gross pathology, or histopathologic findings.

Gene replacement or gene reactivation therapies for sickle-cell disease (SCD) typically target the mutant beta(S)-globin subunits of hemoglobin-S (alpha(2)beta(S)(2)) for substitution by nonpathological beta-like globins. Here we show, in vitro and in vivo in a transgenic mouse model of SCD, that the adverse properties of hemoglobin-S can be reversed by exchanging its normal alpha-globin subunits for zeta-globin, an endogenous, developmentally silenced, non-beta-like globin.

The effect of co-inoculation of a plasmid vector expressing the rabies virus glycoprotein and an additional vector encoding mouse interferon (IFN)-gamma on the development of an antigen specific B and T helper cell response was tested upon intramuscular inoculation of mice. The effect of IFN-gamma was dependent on the promoter driving expression of the viral antigen. The immune responses to antigen-expressing vector carrying a viral promoter such as the SV40 early promoter or the major histocompatibility (MHC) class I promoter were reduced in presence of IFN-gamma while the B and T helper cell response to a vector expressing the antigen under the control of the MHC class II promoter was not affected by this cytokine.

Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.