In a phase 1 dose escalation study, 13 subjects with hemophilia A received by peripheral intravenous infusion a retroviral vector carrying a B-domain-deleted human factor VIII (hFVIII) gene. Infusions were well tolerated. Tests for replication competent retrovirus have been negative. Polymerase chain reaction (PCR) analyses demonstrate the persistence of vector gene sequences in peripheral blood mononuclear cells in 3 of 3 subjects tested. Factor VIII was measured in serial samples using both a one-stage clotting assay and a chromogenic assay. While no subject had sustained FVIII increases, 9 subjects had FVIII higher than 1% on at least 2 occasions 5 or more days after infusion of exogenous FVIII, with isolated levels that ranged from 2.3% to 19%. Pharmacokinetic parameters of exogenous FVIII infused into subjects 13 weeks after vector infusion showed an increased half-life (T 1/2 ; P < .02) and area under the curve (AUC, P < .04) compared with prestudy values. Bleeding frequency decreased in 5 subjects compared with historical rates. These results demonstrate that this retroviral vector (hFVIII(V)) is safe and, in some subjects, persists more than a year in peripheral blood mononuclear cells, with measurable factor VIII levels and with increased available FVIII activity (increased T 1/2 and AUC) after infusion of exogenous FVIII concentrate.

We had previously demonstrated that a cellular protein specifically interacts with the 3 end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D pol , as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1⌬C) diminished these proteinprotein interactions. When GST/hnRNP C1⌬C was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.Picornaviruses effectively subvert host cell functions and efficiently complete their intracellular life cycle in spite of the limited coding capacity of their relatively small single-stranded, positive-sense RNA genomes. Poliovirus (PV), the prototypic member of Picornaviridae, contains a single open reading frame within its 7.4-kb genomic RNA which encodes a 247-kDa polyprotein that is processed by virus-encoded proteinases into numerous intermediate and mature polypeptides. In addition to polyprotein processing, further strategies that successfully expand the biochemical activities encoded in this monocistronic genomic RNA include having distinct activities for precursors and mature polypeptides, embedding multiple activities in a single polypeptide, and recruiting cellular proteins for viral replication functions. After translation and polyprotein processing to generate functional nonstructural viral proteins, replication of positive-sense RNAs occurs via a negative-sense intermediate that ultimately produces new genomes and translation templates for the production of progeny virions. The virus-encoded RNA-dependent RNA polymerase (RdRp), 3D pol , plays a central role in both negative-strand RNA synthesis and in replication of the genomic RNA.Picornavirus researchers have postulated that a cis-acting replication determinant, required for efficient template selection to initiate positive-stra...

We have identified a regulatory region in the human thymidine kinase gene promoter. A set of promoter deletion mutants was constructed, linked to the bacterial neomycin resistance gene, and stably transfected into Rat3 cells. It was shown that the region between 135 and 67 base pairs upstream of the cap site is required for conveying G1-S-phase regulation to the linked neo gene. In addition, primer extension assays demonstrated that the same transcriptional start sites were used in G1- and S-phase cells and in the various deletion mutants tested.

To identify proteins involved in the formation of replication complexes at the 3 end of poliovirus negativestrand RNA, a combined in vitro biochemical and in vivo genetic approach was used. Five subgenomic cDNA constructs were generated to transcribe different negative-strand RNA fragments. In UV cross-linking assays, distinct differences in binding of proteins in extracts from poliovirus-infected and uninfected cells to virusspecific, radiolabeled transcripts were observed. Two proteins present in extracts from poliovirus-infected cells with approximate molecular masses of 36 and 38 kDa were shown to cross-link to the 3 end of poliovirus negative-strand RNA. Appearance of the 36-and 38-kDa proteins in UV cross-linking assays can be detected 3 to 3.5 h after infection, and cross-linking reaches maximum levels by 5 h after infection. The binding site for the 36-kDa protein overlaps with the computer-predicted loop b region of stem-loop I, the so-called cloverleaf structure, and the RNA sequence of this region is required for efficient binding. Transfection of full-length, positive-sense RNA containing a five-nucleotide substitution (positions 20 to 25) in the loop b region of stem-loop I into tissue culture cells yielded only viral isolates with a reversion at position 24 (U3C). This finding demonstrates that the wild-type cytidine residue at position 24 is essential for virus replication. RNA binding studies with transcripts corresponding to the 3 end of negative-strand RNA suggest that complex formation with the 36-kDa protein plays an essential role during the viral life cycle.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.