Newborn screening for Duchenne muscular dystrophy in China: follow-up diagnosis and subsequent treatment.

BACKGROUND: Newborn screening for Duchenne muscular dystrophy (DMD) is currently being initiated in Zhejiang Province, China and is under consideration in other countries, including the United States. As China begins to implement DMD newborn screening (DMD-NBS), there is ongoing discussion regarding the steps forward for follow up care of positively identified patients as well as false positive and false negative results. DATA SOURCES: Relevant papers related to DMD-NBS, and NBS in China were reviewed in PubMed. RESULTS: The current state of DMD-NBS is discussed, along with the steps needed to effectively screen infants for this disease in China, recommendations for establishment of follow up care in patients with positive and negative screens, and measurement of patient outcomes. CONCLUSIONS: Zhejiang Province, China is ready to implement DMD-NBS. Future challenges that exist for this program, and other countries, include the ability to track patients, assist with access to care, and ensure adequate follow-up care according to evidence-based guidelines. In addition, China's large rural population, lack of specialty providers, and difficulty in educating patients regarding the benefits of treatment create challenges that will need to be addressed.

Characterization of a Blood Spot Creatine Kinase Skeletal Muscle Isoform Immunoassay for High-Throughput Newborn Screening of Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive, lethal X-linked neuromuscular disorder with an average worldwide incidence of 1:5000. Blood spot creatine kinase (CK) enzyme assays previously used in newborn screening programs for DMD are nonspecific because measured CK enzyme activity is attributable to 3 isoenzyme forms of CK (CK-MM, CK-MB, and CK-BB) and it is the CK-MM isoform that is found predominantly in skeletal muscle. CK-MM is increased in boys with DMD owing to muscle damage. We describe a sensitive and specific automated immunoassay for CK-MM to screen for DMD in blood spots. METHODS: The prototype assay was developed on the PerkinElmer GSP® analyzer to enable high-throughput screening. CK-MM was assayed using a solid phase, 2-site immunofluorometric system. Purified human CK-MM was used to create calibrators and controls. RESULTS: The limit of blank (LOB), detection (LOD), and quantification (LOQ) values were <1, 3, and 8 ng/mL, respectively. The analytical measurement range was 4-8840 ng/mL. Interassay (n = 40) imprecision was <7% across the analytical range. Cross-reactivity was <5% for CK-MB and 0% for CK-BB. The mean recovery of CK-MM was 101% (range 87%-111%). Blood spots from newborn infants (n = 277) had a mean CK-MM concentration of 155 ng/mL and a 99th centile of 563 ng/mL. The mean blood spot CK-MM concentration from 10 cases of DMD was 5458 ng/mL (range 1217-9917 ng/mL). CONCLUSIONS: CK-MM can be reliably quantified in blood spots. The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD.

Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma.

Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system.

Semliki Forest virus vectors expressing transforming growth factor beta inhibit experimental autoimmune encephalomyelitis in Balb/c mice.

Cytokine immunomodulation of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, has remained a formidable treatment option, but access into the CNS is hampered due to the impermeability of the blood-brain barrier. In this report, we describe the construction and characterization of CNS-homing gene delivery/therapy vectors based on avirulent Semliki Forest virus (SFV) expressing either native or mutant transforming growth factor beta 1 (TGF-beta1). Biological activity of the expressed inserts was demonstrated by PAI-1 promoter driven luciferase production in mink cells and TGF-beta1 mRNA was demonstrated in the CNS of virus treated mice by in situ hybridization and RT-PCR. Both vectors, when given intraperitoneally to EAE mice significantly reduced disease severity compared to untreated mice. Our results imply that immunomodulation by neurotropic viral vectors may offer a promising treatment strategy for autoimmune CNS disorders.