Lentiviral Mediated ADA2 Gene Transfer Corrects the Defects Associated With Deficiency of Adenosine Deaminase Type 2

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood. We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein. In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein. Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).