Comprehensive Preclinical Assessment of ADVM-022, an Intravitreal Anti-VEGF Gene Therapy for the Treatment of Neovascular AMD and Diabetic Macular Edema.

Inhibition of vascular endothelial growth factor is the mode of action for several approved therapies, including aflibercept, for the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME). Lack of compliance due to the frequent intravitreal dosing requirements may result in inadequately treated disease, leading to irreversible vision impairment. To date, the majority of gene therapy clinical trials providing sustained anti-VEGF levels in the retina have been limited to subretinal injections requiring a vitrectomy.

Long-Term Safety Evaluation of Continuous Intraocular Delivery of Aflibercept by the Intravitreal Gene Therapy Candidate ADVM-022 in Nonhuman Primates.

Purpose: To evaluate the long-term safety of vascular endothelial growth factor (VEGF) suppression with sustained aflibercept expression after a single intravitreal injection (IVI) of ADVM-022, an anti-VEGF gene therapy, in non-human primates (NHPs).

Methods: Non-human primates received bilateral IVI of ADVM-022, a gene therapy vector encoding aflibercept, a standard of care for the treatment of VEGF-based retinal disease. Aflibercept levels from ocular fluids and tissues were measured.

Analysis of Aflibercept Expression in NHPs following Intravitreal Administration of ADVM-022, a Potential Gene Therapy for nAMD.

Several standard-of-care therapies for the treatment of retinal disease, including aflibercept, inhibit vascular endothelial growth factor (VEGFA). The main shortcoming of these therapies is potential undertreatment due to a lack of compliance resulting from the need for repeated injections. Gene therapy may provide sustained levels of anti-VEGFA proteins in the retina following a single injection.

Preclinical Evaluation of ADVM-022, a Novel Gene Therapy Approach to Treating Wet Age-Related Macular Degeneration.

Inhibition of vascular endothelial growth factor, a key contributor to the choroidal neovascularization associated with wet age-related macular degeneration, is the mode of action of several approved therapies, including aflibercept, which requires frequent intravitreal injections to provide clinical benefit. Lack of compliance with the dosing schedule may result in recurrence of active wet macular degeneration, leading to irreversible vision impairment. Gene therapy providing sustained anti-vascular endothelial growth factor levels in the retina following a single injection could drastically reduce the treatment burden and improve visual outcomes.

Engineering goals for future thoracic endografts-how can we make them more effective?

Endovascular treatments for catastrophic aortic conditions have gained increasing popularity over the past 20 years. Originally developed for abdominal aortic aneurysms (EVAR), treatment has been modified for use in thoracic aortic repair (TEVAR). As expanding numbers of patients with increasingly intractable conditions and more hostile anatomies are treated, endovascular stent designs are maturing to be suitable for these more demanding situations.

American Society of Hematology--40th annual meeting. 4-8 December 1998, Miami Beach, FL, USA.

The annual meeting of the American Society of Hematology was attended by over 30,000 researchers, nurses, hematologists and physicians. The work presented covered a spectrum from basic science, drugs in development and reports of clinical trials. Over 4600 abstracts were submitted with 3000 presented mainly in poster form.

Factors affecting long-term expression of a secreted transgene product after intravenous administration of a retroviral vector.

We have studied parameters affecting in vivo expression of human growth hormone (hGH) in mice after intravenous administration of a retroviral vector encoding the protein as a model system for clotting factor VIII gene therapy. Such treatment results in a brief burst of high-level expression followed by lower level sustained expression of the hGH in the circulation. The major targets for transduction in the mouse are liver and spleen.

Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction.

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V).

Genetic induction of a releasable pool of factor VIII in human endothelial cells.

Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release is less clear. Previously, we showed that FVIII can traffic together with von Willebrand factor (vWF) into storage granules in a pituitary tumor cell line, AtT-20; however, AtT-20 cells could not be used to address the release or functional activity of released FVIII. To investigate the regulated secretion of stored FVIII, endothelial cells with intact agonist-stimulated release pathways were used.

Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer.

For many applications, human clinical therapies using retroviral vectors still require many technological improvements in key areas of vector design and production. These improvements include higher unprocessed manufacturing titers, complement-resistant vectors, and minimized potential to generate replication-competent retrovirus (RCR). To address these issues, we have developed a panel of human packaging cell lines (PCLs) with reduced homology between retroviral vector and packaging components.

The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F.

Venous thromboembolism associated with double heterozygosity for R506Q mutation of factor V and for T298M mutation of protein C in a large family of a previously described homozygous protein C-deficient newborn with massive thrombosis.

It is remarkable that certain patients with heterozygous protein C (PC) deficiency manifest venous thromboembolism (VTE), whereas others, particularly those belonging to families with homozygous PC deficiency, remain asymptomatic. The goals of the present study of a family, in which the proband had homozygous PC deficiency, were to identify members with and without VTE, to determine the mutation causing PC deficiency, and to search for the R506Q mutation of factor V (FV) causing activated PC resistance. Heterozygosity for a T298M mutation in exon 9 of the PC gene was found in the father of the homozygous proband who died of massive thrombosis.

A two-allele polymorphism in protein C inhibitor with varying frequencies in different ethnic populations.

cDNAs for protein C inhibitor (PCI), prepared from human liver RNA, contained two forms of PCI, designated PCI*A and PCI*B. While PCI*A is identical to the published PCI sequence, PCI*B differs in 4 of 1221 bp and two amino acids, A36V and K86E. Frequencies for the PCI*B allele, determined from genomic DNA, differed among ethnic groups.

Two mutations in the promoter region of the human protein C gene both cause type I protein C deficiency by disruption of two HNF-3 binding sites.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by the proteolytic inactivation of factors Va and VIIIa. Individuals affected with protein C deficiency are at risk for thrombosis. Genetic analyses of affected individuals, to determine the cause of the protein C deficiency, revealed a large variety of mutations in the protein C gene, including several in the promoter region of this gene.

Characterization of a cDNA for rhesus monkey protein C inhibitor--evidence for N-terminal involvement in heparin stimulation.

cDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues.

Activated protein C resistance: molecular mechanisms.

Activated protein C (APC) resistance is usually associated with a single DNA mutation predicting replacement of Arg506 by Gln in factor V (FV). Studies using synthetic peptides suggest that FV residues 493-506 provide factor Xa (FXa) and protein S binding sites. Biochemical studies were performed to test the hypothesis that the Arg506Gln FV mutation causes APC resistance and to define the nature of the resistance of Gln506-FVa to APC.

Incidence of activated protein C resistance caused by the ARG 506 GLN mutation in factor V in 113 unrelated symptomatic protein C-deficient patients. The French Network on the behalf of INSERM.

Because multiple risk factors in one patient may increase the clinical expression of thrombophilia, we assessed the presence in protein C-deficient patients of the factor V Arg 506 Gln mutation responsible for activated protein C resistance. Using a strategy allowing rapid screening of factor V exon 10, we studied 113 patients with protein C deficiency and 104 healthy volunteers. We detected the Arg 506 Gln mutation in 15 patients (14%) and in one healthy subject (1%).

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V.

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity.

Use of a generally applicable tissue factor--dependent factor V assay to detect activated protein C-resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant.

The original activated partial thromboplastin time-based assay for activated protein C (APC)-resistant factor Va (FVa) requires carefully prepared fresh plasma and cannot be used in patients receiving warfarin or in patients with antiphospholipid antibodies. A new test is described here that circumvents these limitations and distinguishes without overlap heterozygotes for APC-resistant FVa from persons with normal FV. A diluted test plasma is incubated with an FV-deficient substrate plasma and tissue factor and then clotted with Ca2+ or Ca2+ plus APC.

Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C.

Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active.

Anticoagulant response to activated protein C: method validation and assay comparison.

Poor anticoagulant response to activated protein C (APC), present in 20-60% of thrombophilic patients, is most often caused by abnormal factor Va due to the mutation of Arg506 to Gln, and DNA sequencing confirms this finding. At Scripps Reference Laboratory (SRL), we have validated an in-house assay to detect APC resistance. A study of 80 normal subjects (40 males and 40 females, 21-60 years old) showed that adult males and females have statistically significant differences in their anticoagulant response to APC.