Arthur W. Nienhuis

Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B

BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major

BACKGROUND To determine whether deferoxamine prevents the complications of transfusional iron overload in thalassemia major, we evaluated 59 patients (30 were female and 29 male; age range, 7 to 31 years) periodically for 4 to 10 years or until death. METHODS At each follow-up visit, we performed a detailed clinical and laboratory evaluation and measured hepatic iron stores with a noninvasive magnetic device. RESULTS The body iron burden as assessed by magnetic measurement of hepatic iron stores was closely correlated (R = 0.89, P < 0.001) with the ratio of cumulative transfusional iron load to cumulative deferoxamine use (expressed in millimoles of iron per kilogram of body weight, in relation to grams of deferoxamine per kilogram, transformed into the natural logarithm). Each increase of one unit in the natural logarithm of the ratio (transfusional iron load to deferoxamine use) was associated with an increased risk of impaired glucose tolerance (relative risk, 19.3; 95 percent confidence interval, 4.8 to 77.4), diabetes mellitus (relative risk, 9.2; 95 percent confidence interval, 1.8 to 47.7), cardiac disease (relative risk, 9.9; 95 percent confidence interval, 1.9 to 51.2), and death (relative risk, 12.6; 95 percent confidence interval, 2.4 to 65.4). All nine deaths during the study occurred among the 23 patients who had begun chelation therapy later and used less deferoxamine in relation to their transfusional iron load (P < 0.001). CONCLUSIONS The early use of deferoxamine in an amount proportional to the transfusional iron load reduces the body iron burden and helps protect against diabetes mellitus, cardiac disease, and early death in patients with thalassemia major.

Pure red-cell aplasia of 10 years' duration due to persistent parvovirus B19 infection and its cure with immunoglobulin therapy

PARVOVIRUS B19 is the etiologic agent of transient aplastic crisis, an acute episode of bone marrow failure in persons with underlying hemolysis.1 , 2 In tissue-culture studies, parvovirus B19 infe...

Localization of the human α-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

Abstract We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.

Long-term Safety and Efficacy Following Systemic Administration of a Self-complementary AAV Vector Encoding Human FIX Pseudotyped With Serotype 5 and 8 Capsid Proteins

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.

Vitamin C and Iron

Some of the first clues that ascorbic acid might be involved in iron metabolism came from careful clinical observations of the Bantu in South Africa.1 Habituated to the practice of drinking large q...

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Identification of a stage selector element in the human gamma-globin gene promoter that fosters preferential interaction with the 5' HS2 enhancer when in competition with the beta-promoter.

The erythroid‐specific enhancer within hypersensitivity site 2 (HS2) of the human beta‐globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the gamma‐ and beta‐promoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The beta‐promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a gamma‐promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the gamma‐promoter for HS2 included those between positions −53 and −35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the beta‐promoter, increased beta‐promoter activity 10‐fold when linked to HS2. The modified beta‐promoter was also capable of competing with a gamma‐promoter modified internally in the −53 to −35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago gamma‐promoter, a species which lacks fetal gamma‐gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the −53 to −35 sequence of the gamma‐promoter. We speculate that this region of the gamma‐promoter functions as a stage selector element in the regulation of hemoglobin switching in humans.

Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein.

The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma‐globin promoter is integral to the competitive silencing of a linked beta‐promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti‐CP2 antiserum disrupts the formation of the SSP‐SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40‐45 kDa in UV and protein cross‐linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A‐gene‐promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF‐E4. Comparative studies between human CP2 and chicken NF‐E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti‐CP2 antiserum or a monoclonal antibody to NF‐E4. Western analysis of partially purified NF‐E4 using anti‐CP2 antiserum or the NF‐E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF‐E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP‐SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

USE OF THE GREEN FLUORESCENT PROTEIN AS A MARKER TO IDENTIFY AND TRACK GENETICALLY MODIFIED HEMATOPOIETIC CELLS

Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells