Ex vivo gene delivery to synovium using foamy viral vectors.

BACKGROUND: Gene transfer technologies have the potential to fundamentally improve current therapies for arthritic conditions, although this is essentially dependent on safe and efficient vector systems. The foamy virus (FV)-based vectors have many safety features that favour their use in the treatment of arthritis. In the present study, we investigated the use of safe prototype foamy viral vectors (FVV) for indirect gene delivery to articular tissues. METHODS: We generated recombinant FVV encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA under the control of the spleen focus forming virus U3 promoter and explored their transgene expression profile following ex vivo gene delivery to knee joints of Wistar and athymic nude rats. RESULTS: FVV efficiently transduced primary rat synovial fibroblasts using the EGFP and the IL1RA transgene in vitro. FVV-mediated IL1RA expression was functional in blocking IL1 effects in vitro. After the transplantation of FVV transduced synovial fibroblasts, the intra-articular transgene expression in Wistar rats was initially high and declined after approximately 3 weeks for both transgenes. By contrast, FVV-mediated expression of EGFP and IL1RA persisted for at least 12 weeks at high levels in immunocompromised nude rats. FVV-meditated gene delivery was well tolerated by all animals without extra-articular transgene expression, arguing for the safety of this approach. CONCLUSIONS: Our results indicate that FVV are capable of efficient ex vivo gene transfer to synovium and merit further investigation as a means to provide long-term intra-articular transgene expression for arthritis treatment.

Foamy virus-adenovirus hybrid vectors for gene therapy of the arthritides.

BACKGROUND: Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. METHODS: We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. RESULTS: FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10-35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3-5 days after injection of FAD vectors to the knee joints. CONCLUSIONS: Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides.

Heparan sulfate is an attachment factor for foamy virus entry.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

[The importance of the underlying disease for outcome of patients with implanted automatic defibrillators]. / Die Bedeutung der Grunderkrankung für den Krankheitsverlauf bei Patienten mit implantiertem automatischen Defibrillator.

It is unclear whether the outcome of patients with implanted cardioverter defibrillator (ICD) is influenced by the underlying etiology or not. Therefore, we studied the follow-up of 271 patients who underwent ICD implantation for life-threatening ventricular tachyarrhythmias. Coronary artery disease was present in 203 patients (75%) (G1), dilated cardiomyopathy in 36 patients (18%) (G2), while 32 patients (12%) (G3) had an "arrhythmogenic" ventricle (dysplasia, valvular disease, idiopathic arrhythmias). Mean left ventricular ejection fraction was 30 +/- 11% in G1, 33 +/- 13% in G2 and 48 +/- 13% in G3. Perioperatively, 12/271 patients (4%) died. During the mean follow-up of 21 +/- 17 (< 1 to 99) months, 52/259 patients (20%) died: 31% (11/36 patients) in G2, 19% (36/193 patients) in G1 and 17% (5/39 patients) in G3. There was a low incidence of sudden death (SD) (4%, 2% per year) without significant differences between G1 (3%), G2 (8%) and G3 (3%). In addition, no significant differences were observed in cardiac mortality (CD) between G1 (10%), G2 (14%) and G3 (3%) (p = n.s.). ICD discharges occurred in 188 patients (69%); ICD discharges occurred in G1 in 138 patients (68%), in G2 in 27 patients (75%) and in G3 in 23 patients (72%). The mean incidence of ICD discharges per patient was 16 +/- 10 shocks in G1, 24 +/- 8 shocks in G2 and 18 +/- 8 shocks in G3. After ICD implant, complications occurred in 67 patients (23%). Our data show that the ICD is highly effective in preventing sudden death independent on the underlying etiology.(ABSTRACT TRUNCATED AT 250 WORDS)

[Results and experiences of transvenous endocardial defibrillator therapy]. / Ergebnisse und Erfahrungen der transvenösen endokardialen Defibrillatortherapie.

We studied the follow-up of 72 patients who underwent implantation of a transvenous defibrillation lead system (ELS) (Endotak, CPI). All patients had ventricular tachycardia (VT) or fibrillation (VF) refractory to antiarrhythmic drug therapy. There were 51 patients with coronary disease and 21 patients had non-ischemic VT/VF. ELS was combined with a subcutaneous patch in 52 patients and implanted alone ("single lead only") in 20 patients. 40 patients received the ELS combined with antitachycardia pacing devices (Ventak PRx, CPI; Cadence, Ventritex) and 32 patients with the Ventak P 1600 or P2, CPI. Implantation of the ELS was attempted in 80 patients and performed in 72 patients (90%): Intraoperatively, the mean defibrillation threshold (DTF) was > 25 Joule (J) in five patients and no reliable ELS position was possible to achieve in three patients. These eight patients underwent thoracotomy with epicardial patch implantation. The mean DFT was < or = 20 J in all 72 patients with a mean DFT of 14 +/- 8 J in VT patients and 17 +/- 10 J in VF patients. Two of 80 patients (3%) died: one patient died intraoperatively and one during the mean follow-up of 6 +/- 2 (< 1 to 18) months. Complications occurred in three patients (4%): Dislocation of the Endotak electrode was observed in two patients (3%) and one patient developed pneumothorax postoperatively. Our data show that the ELS is most suitable in the majority of patients with VT/VF and is the approach of first choice for cardioverter defibrillator implantation at the present time. However, despite a relatively low intra- and perioperative complication rate, this approach should not be performed in institutions without cardiac surgery.