X-linked adrenoleukodystrophy (ALD) is a severe brain demyelinating disease in boys that is caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. ALD progression can be halted by allogeneic hematopoietic cell transplantation (HCT). We initiated a gene therapy trial in two ALD patients for whom there were no matched donors. Autologous CD34+ cells were removed from the patients, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1, and then re-infused into the patients after they had received myeloablative treatment. Over a span of 24 to 30 months of follow-up, we detected polyclonal reconstitution, with 9 to 14% of granulocytes, monocytes, and T and B lymphocytes expressing the ALD protein. These results strongly suggest that hematopoietic stem cells were transduced in the patients. Beginning 14 to 16 months after infusion of the genetically corrected cells, progressive cerebral demyelination in the two patients stopped, a clinical outcome comparable to that achieved by allogeneic HCT. Thus, lentiviral-mediated gene therapy of hematopoietic stem cells can provide clinical benefits in ALD.

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.The retroviral life cycle requires that significant amounts of RNA remain unspliced and perform several functions in the cytoplasm. Thus, the full-length RNA serves as the viral genetic material that will be encapsidated in viral particles and as the mRNA encoding structural and enzymatic proteins required for viral replication. Simple retroviruses are defined as those viruses which produce one single-spliced env RNA from this full-length precursor RNA, whereas complex retroviruses are characterized by the production of multiple spliced RNA species (18). Because cis-acting and coding functions in the viral genome frequently overlap, most of the studies on RNA splicing and transport regulation in murine leukemia viruses (MuLV) have been conducted with extensively reshaped retroviral vectors that are replication defective (9,14,17). Besides these models with vectors, usage of MuLV canonical or cryptic splice sites has been reported in the context of insertional mutagenesis occurring in animals inoculated with the replication-competent wild-type Moloney MuLV (2,15,19,22,26). Since such events are accompanied by the rearrangement of both the target cellular proto-oncogene and the inserted proviral DNA (20,26), the mechanisms controlling the differential usage of the splice sites remain unclear.In the present work, performed on cells infected ex vivo with the closely related replication-competent Friend and Moloney MuLVs, we report for the first time the production of a large subgenomic transcript in a simple retrovirus. This novel RNA was generated from an alternative splice donor site, SDЈ, which is conserved among mammalian simple retroviruses, in combination with the env splice acceptor site. Mutations of SDЈ in Friend and Moloney MuLVs affect the general splicing pattern of viral RNAs and reduce viral replication. Identification of this novel MuLV subgenomic RNA provides new insights into simple retrovirus functions. MATERIALS AND METHODSMutagenesis and proviral constructs. The parental prototypic Friend MuLV strain 57 (16) and Moloney MuLV strain 8.2 (23) were used as permutated molecular clones with a single copy of the long terminal repeat (24). These clones provided the wild-type (wt) background sequences for the reconstitution of replication-competent Friend and Moloney mutants. Mutations were introduced by PCR with oligonucleotide-directed mutagenesis (11). The pMSD1/B plasmid carrying the entire Moloney MuLV sequence with the M1 mutation was pre...