Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SUMMARY: Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

Volumetric modulated arc therapy with simultaneous integrated boost for locally advanced rectal cancer.

AIMS: To report the feasibility of volumetric modulated arc therapy (VMAT) for neoadjuvant radiotherapy in locally advanced rectal cancer in a dose-escalation protocol and simultaneous integrated boost (SIB) approach. Moreover, the VMAT technique was compared with three-dimensional conformal radiotherapy (3D-CRT) and fixed-field intensity modulated radiotherapy (IMRT), in terms of target coverage and irradiation of organs at risk. MATERIALS AND METHODS: Eight patients with locally advanced rectal cancer were treated with the SIB-VMAT technique. The VMAT plans were compared with 3D-CRT and IMRT techniques in terms of several clinically dosimetric parameters. The number of monitor units and the delivery time were analysed to score the treatment efficiency. All plans were verified in a dedicated solid water phantom using a two-dimensional array of ionisation chambers. RESULTS: All techniques meet the prescription goal for planning target volume coverage, with VMAT showing the highest level of conformality. VMAT is associated with 40, 53 and 58% reduction in the percentage of volume of small bowel irradiated to 30, 40 and 50Gy, compared with 3D-CRT. No significant differences were found with respect to SIB-IMRT. VMAT plans showed a significant reduction of monitor units by nearly 20% with respect to IMRT and reduced treatment time from 14 to 5min for a single fraction. CONCLUSIONS: SIB-VMAT plans can be planned and carried out with high quality and efficiency for rectal cancer, providing similar sparing of organs at risk to SIB-IMRT and resulting in the most efficient treatment option. SIB-VMAT is currently our standard approach for radiotherapy of locally advanced rectal cancer.

Real-time dose reconstruction for wedged photon beams: a generalized procedure.

A practical and accurate generalized procedure to reconstruct the isocenter dose D(iso) for 3D conformal radiotherapy (3DCRT) has been developed for X-ray open beams supplied by linacs of different manufacturers and equipped with aSi electronic portal imaging devices (aSi EPIDs). This paper reports an extension of the method, to be applied at the wedged X-ray beams characterized by the wedge attenuation factor W(AF). Using water-equivalent solid phantoms (SPs) of different thicknesses, w, and photon square fields of sizes, L, the generalized midplane doses D(0)(W(AF), w/2,L) and generalized transit signals s(t)(0)(W(AF),w,L) by 38 beams of six different linacs were determined. The generalized data were fitted by surface equations and used together with the information of the 'record & verify' network of the centers. In this manner, for every beam, the D(iso) reconstruction was obtained in about 25 seconds after the treatment. To test the in vivo dosimetric procedure, six pelvic treatments that used conformed wedged beams were carried out with three linacs of different manufacturers. For every beam, the comparison between the reconstructed D(iso) and the D(iso,TPS) computed by the TPS, resulted in an acceptable tolerance level of ±5%, estimated for this kind of treatment. Generally the in vivo dosimetry methods that use EPIDs require: (i) a special effort for the dosimetric commissioning with SPs of different thicknesses, and (ii) extra time for the analysis of the EPID signals. The proposed procedure simplifies the commissioning step and supplies for Varian, Elekta, and Siemens linacs equipped with the aSi EPIDs a quasi-real time in vivo dosimetry for open and wedged 3DCRT fields.

Calibration of Elekta aSi EPIDs used as transit dosimeter.

The transit in vivo dosimetry performed by the Electronic Portal Imaging Device (EPID), avoids the problem of solid-state detector positioning on the patient. Moreover, the dosimetric characterization of the recent Elekta aSi EPIDs in terms of signal stability and linearity enables these detectors adaptable for the transit in vivo dosimetry with 6, 10 and 15 MV photon beams. However, the implementation of the EPID transit dosimetry requires several measurements. Recently, the present authors have developed an in vivo dosimetry method for the 3D CRT based on correlation functions defined by the ratios between the transit signal, s(t) (w,L), by the EPID and the phantom mid-plane dose, D(m)(w,L), at the Source to Axis Distance (SAD) as a function of the phantom thickness, w, and the square field dimensions, L. When the phantom mid-plane was positioned at distance d from the SAD, the ratios st(w,L)/s't(d,w,L), were used to take into account the variation of the scattered photon contributions on the EPID as a function of, d and L. The aim of this paper was the implementation of a procedure that uses generalized correlation functions obtained by nine Elekta Precise linac beams. The procedure can be used by other Elekta Precise linacs equipped with the same aSi EPIDs assuring the stabilities of the beam output factors and the EPID signals. The calibration procedure of the aSi EPID here reported avoids measurements in solid water equivalent phantoms needed to implement the in vivo dosimetry method in the radiotherapy center. A tolerance level ranging between ±5% and ±6% (depending on the type of tumor) was estimated for the comparison between the reconstructed isocenter dose, D(iso) and the computed dose D(iso,TPS) by the treatment planning system (TPS).

Solid and papillary epithelial neoplasm of the pancreas in an 11-year-old girl: case report and literature review.

An 11-year-old girl presented with episodic abdominal pain of 2 years' duration. CT scan of the abdomen showed a mass in the tail of the pancreas. A distal pancreatectomy was done and the tumor was excised. Macroscopic and immunohistochemical studies were compatible with a solid and papillary epithelial neoplasm. This is a rare neoplasm with a decidedly female predominance. It has a very low malignant potential with a good prognosis. Surgical removal of the tumor is usually curative.

Optimized production of high-titer recombinant adeno-associated virus in roller bottles.

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.

Development of a stable retrovirus vector capable of long-term expression of gamma-globin mRNA in mouse erythrocytes.

Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human gamma-globin gene showed position-independent, copy number-dependent expression of a linked gamma-globin mRNA. We generated a "double-copy" Ank/A gamma-globin retrovirus vector that transferred two copies of the Ank/A gamma-globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long-term repopulating hematopoietic stem cells. Expression of Ank/A gamma-globin mRNA in mature red blood cells was approximately 8% of the level of mouse alpha-globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy.

As initial human gene therapy trials for beta-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve beta-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe beta-thalassemia. Generation of beta-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, beta-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human gamma-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous alpha-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, gamma-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total alpha-globin mRNA in patients with severe beta-thalassemia in whom 20% of erythroid precursors express the vector genome.

Long-term expression of gamma-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma-globin gene fused to the ankyrin-1 promoter.

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked gamma-globin gene in transgenic mice. We inserted the Ank/(A)gamma-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/(A)gamma-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/(A)gamma-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse alpha-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe beta-thalassemia if the level of expression can be further increased.

Cobalamin deficiency presenting with cutaneous hyperpigmentation: a report of two siblings.

The authors report on 2 children with pernicious anemia, sisters, who presented with hypermelanosis as one of the clinical manifestations. The hypermelanosis disappeared with adequate treatment of vitamin B12 deficiency. Vitamin B12 deficiency should be considered in the differential diagnosis of a child presenting with hyperpigmentation and macrocytic red cell indices.

Amphotropic or gibbon ape leukemia virus retrovirus binding and transduction correlates with the level of receptor mRNA in human hematopoietic cell lines.

The low level of amphotropic retrovirus mediated gene transfer into human hematopoietic stem cells (HSC) has been an impediment to gene therapy for hematopoietic diseases (1). We have previously shown that mouse and human HSC have low levels of the mRNA encoding PiT-2, the amphotropic retrovirus receptor. We hypothesized that the low level of PiT-2 mRNA was responsible for the low frequency of transduction of HSC by amphotropic retroviral vectors (2). In this study we compared the level of PiT-2 and PiT-1, the Gibbon Ape Leukemia Virus receptor (GaLV), in 5 human tissue culture cell lines. PiT-2 and PiT-1 mRNA levels were highest in K562 cells and lowest in HL60 cells. In hematopoietic cell lines, the level of PiT-2 or PiT-1 mRNA correlated directly with retrovirus binding and transduction with the appropriate (amphotropic or GaLV) retrovirus vector. The level of expression of PiT-2 and PiT-1 mRNA could be increased by treatment of HL60 cells with either PMA or Interleukin-1alpha. The increase in the level of PiT-2 and PiT-1 mRNA correlated with increased transduction with both amphotropic and GaLV retroviral vectors. We conclude that the improved transduction was a direct effect of the increased levels of receptor mRNA and unrelated to changes in the cell cycle status.

Simultaneous pulmonary leiomyosarcoma and leiomyoma in pediatric HIV infection.

The case of a 7-year-old girl with acquired immunodeficiency syndrome treated for 5 years with AZT and intravenous gamma globulin is reported. Shortly before her demise she developed a pulmonary leiomyosarcoma and leiomyoma. Does prolonged survival in pediatric acquired immune deficiency syndrome increase the incidence of secondary malignancies?

Plummer-Vinson syndrome in an adolescent.

We report an 18-year-old female found to have Plummer-Vinson syndrome on routine health evaluation. The nature of this condition, its relation to iron deficiency, and its potential for malignancy are discussed.