Cystic fibrosis, sickle-cell anemia could be corrected in embryos with new CRISPR variant.


Since the discovery of the genome-editing tool CRISPR/Cas9, scientists have been looking to utilize the technology to make a significant impact on correcting genetic diseases. Technical challenges have made it difficult to use this method to correct disorders that are caused by single-nucleotide mutations, such as cystic fibrosis, sickle-cell anemia, Huntington’s disease, and phenylketonuria. … [Researchers] have just used a variation of CRISPR/Cas9 to produce mice with single-nucleotide differences. The findings from this new study were published recently in Nature Biotechnology in an article entitled “Highly Efficient RNA-Guided Base Editing in Mouse Embryos.

The most frequently used CRISPR/Cas9 technique works by cutting around the faulty nucleotide in both strands of the DNA and cuts out a small part of DNA. In the current study, the investigators used a variation of the Cas9 protein (nickase Cas9, or nCas9) fused with an enzyme called cytidine deaminase, which can substitute one nucleotide into another—generating single-nucleotide substitutions without DNA deletions

“The next goal is to correct a genetic defect in animals. Ultimately, this technique may allow gene correction in human embryos,” [remarked senior study investigator Jin-Soo Kim].

Cystic Fibrosis: New Hope for Gene Therapy?


Gene therapy to treat cystic fibrosis patients was associated with stabilization — but not improvement — in lung function, according to the results of a randomized, double-blinded phase IIb trial of the therapy in the U.K.

In a per protocol analysis, Professor Eric W.F.W. Alton, of Imperial College in London, and colleagues found a significant, but modest, treatment effect in cystic fibrosis patients treated with the nonviral, chemically designed gene-liposome pGM169/GL67A compared with those treated with a placebo of 0.9% saline (3.7%, 95% CI 0.1-7.3, P=0.046).

While a significant ANCOVA-adjusted effect was observed after 12 months’ follow-up, relative differences in FEV1 after 12 months of treatment were -0.4% (95% CI -2.8 to 2.1) for the treatment group compared to -4.0% (CI -6.6 to -1.4) for the placebo, they reported in The Lancet Respiratory Medicine.

The primary endpoint of the study was defined as relative percent change in forced expiratory volume (FEV1) after 12 months. The authors achieved that because while treatment with pGM169/GL67A did not improve lung function, it did not worsen it either.

Modest Benefit

“This study proves for the first time that copies of the normal CF gene delivered by aerosol inhalation can have a measurable beneficial effect on lung function, compared with placebo, in patients with cystic fibrosis,” senior co-author Dr. Alastair Innes, of Western General Hospital in Edinburgh, Scotland, told MedPage Today. He added that while the effect was statistically significant, he also described it as “modest.”

A small number of patients in both groups did experience improvements in lung function. The authors note that a post-hoc analysis showed 18% of patients (15 in the treatment group and six in the control group) showed an improvement in percent predicted FEV1 of 5% or more of their initial baseline values. By contrast, overall treatment effect in the 65 patients in the treatment group and 56 in the control group was 3.6% (95% CI 0.2-7.0,P=0.039). Of the 20 patients who did not complete the full treatment of one dose per 28 days for 12 months, they received a mean 3.7 doses (SD 1-9).

Patients were randomized into a number of stratified subgroups, but the authors attributed any treatment effect to a greater decline in FEV1 from the placebo group as opposed to greater improvement from pGM169/GL67A. Stratifying by baseline predicted FEV1 (<70% versus ≥70%) found that patients with a more severe disease (FEV1 49.6%-69.2% predicted) had a treatment effect of 6.4% (95% CI 0.8-12.1). By contrast, those with less severe disease (FEV1 69.6%-89.9% predicted) had a 0.2% treatment effect (CI -4.6 to 4.9, P interaction=0.065).

The authors also cited the post-trial and pre-trial changes in the placebo group (-4.9%) compared with the treatment group (1.5%) as contributing to the treatment effect. There were no differences observed by age, sex, or CTFR mutation.

The study also had a number of secondary outcomes, which achieved mixed results. Patients in the treatment group experienced greater improvements in forced vital capacity (FVC) and CT gas trapping, or the inability to exhale completely (P=0.031 andP=0.048, respectively) than the control group. But authors observed no treatment effect for other measures of lung function, imaging, and quality of life. Similar to the primary analysis, they did note that secondary outcomes tended to be more favorable for those with more severe disease.

The authors commented that patients with more severe disease seemed to experience an enhanced treatment effect, and saw this as an opportunity for further research.

“A larger trial with a stratified trial entry design, powered to assess subgroups, and that addresses the mechanisms of response heterogeneity will be important to verify or refute these data,” they wrote.

A total of six serious adverse events were recorded from the pGM169/GL67A group. The committee judged that they were unrelated to the treatment, though one may have been related to a trial procedure (bronchoscopy), the authors said. Two patients total discontinued treatment; one in the placebo group due to fatigue and one in the treatment group due to flu-like symptoms. There were no deaths during the study, and the authors saw no clinically relevant changes in patients throughout the study.

This randomized, double-blinded, placebo-controlled trial consisted of two cystic fibrosis centers in London and Edinburgh at 18 sites in the U.K. from June 12, 2012, to June 24, 2013. Participants were eligible if they were ages ≥12 years, had a FEV1 of 50%-90% predicted and had any combination of CFTR gene mutations. Of the 140 patients, 78 received pGM169/GL67A and 62 received a placebo. There were 116 patients (83%) completing the treatment and included in the per protocol analysis.

Limitations

The most important limitation the authors cite is that the mean difference is at the lower end of clinical trials for gene therapy in patients with cystic fibrosis, mainly due to the reduction in FEV1 volume in the placebo group. They suggest several reasons for this, such as optimal respiratory health for patients at time of trial entry, enthusiasm for the trial leading to improvements in lung function during the recruitment period, and that the trial included all available data, even if the patients were unstable, while registry data only contains measurements from an annual review. They also note the trial’s heterogeneous response and that the fact that changes may be the result of a “non-specific response” to the pGM169/GL67A treatment.

The authors describe their conclusions as a “proof of concept” for nonviral CFTR gene therapy, calling it “another step along the path of translational cystic fibrosis gene therapy.”

Innes said that the efficiency of the gene uptake needs to be improved before the therapy is applicable to clinical practice, adding that the UK Gene Therapy Consortium is engaged in pursuing several lines of research.

“We are exploring whether increased or more frequent dosing would increase benefit, the possible additional benefit of combining gene therapy with other basic treatments which help the CF ion channel to remain open, and novel viral gene therapy vectors which may increase the efficiency of gene transfer,” he said.

Yale scientists use gene editing to correct mutation in cystic fibrosis


Left to right, cystic fibrosis cells treated with gene-correcting PNA/DNA show increasing levels of uptake, or use to correct the mutation. (Images by Rachel Fields)

Yale researchers successfully corrected the most common mutation in the gene that causes cystic fibrosis, a lethal genetic disorder.

The study was published April 27 in Nature Communications.

Cystic fibrosis is an inherited, life-threatening disorder that damages the lungs and digestive system. It is most commonly caused by a mutation in the cystic fibrosis gene known as F508del. The disorder has no cure, and treatment typically consists of symptom management. Previous attempts to treat the disease through gene therapy have been unsuccessful.

To correct the mutation, a multidisciplinary team of Yale researchers developed a novel approach. Led byDr. Peter Glazer, chair of therapeutic radiology, Mark Saltzman, chair of biomedical engineering, and Dr. Marie Egan, professor of pediatrics and of cellular and molecular physiology, the collaborative team used synthetic molecules similar to DNA — called peptide nucleic acids, or PNAs — as well as donor DNA, to edit the genetic defect.

“What the PNA does is clamp to the DNA close to the mutation, triggering DNA repair and recombination pathways in cells,” Egan explained.

The researchers also developed a method of delivering the PNA/DNA via microscopic nanoparticles. These tiny particles, which are billionths of a meter in diameter, are specifically designed to penetrate targeted cells.

In both human airway cells and mouse nasal cells, the researchers observed corrections in the targeted genes. “The percentage of cells in humans and in mice that we were able to edit was higher than has been previously reported in gene editing technology,” said Egan. They also observed that the therapy had minimal off target, or unintended, effects on treated cells.

While the study findings are significant, much more research is needed to refine the genetic engineering strategy, said Egan. “This is step one in a long process. The technology could be used as a way to fix the basic genetic defect in cystic fibrosis.”

Blood mRNA biomarkers for detection of treatment response in acute pulmonary exacerbations of cystic fibrosis.


Abstract

Background Acute pulmonary exacerbations accelerate pulmonary decline in cystic fibrosis (CF). There is a critical need for better predictors of treatment response.

Objective To test whether expression of a panel of leucocyte genes directly measured from whole blood predicts reductions in sputum bacterial density.

Methods A previously validated 10-gene peripheral blood mononuclear cell (PBMC) signature was prospectively tested in PBMC and whole blood leucocyte RNA isolated from adult subjects with CF at the beginning and end of treatment for an acute pulmonary exacerbation. Gene expression was simultaneously quantified from PBMCs and whole blood RNA using real-time PCR amplification. Test characteristics including sensitivity, specificity, positive and negative predictive values were calculated and receiver operating characteristic curves determined the best cut-off to diagnose a microbiological response. The findings were then validated in a smaller independent sample.

Results Whole blood transcript measurements are more accurate than forced expiratory volume in 1 s (FEV1) or C reactive protein (CRP) alone in identifying reduction of airway infection. When added to FEV1, the whole blood gene panel improved diagnostic accuracy from 64% to 82%. The specificity of the test to detect reduced infection was 88% and the positive predictive value for the presence of persistent infection was 86%. The area under the curve for detecting treatment response was 0.81. Six genes were the most significant predictors for identifying reduction in airway bacterial load beyond FEV1 or CRP alone. The high specificity of the test was replicated in the validation cohort.

Conclusions The addition of blood leucocyte gene expression to FEV1 and CRP enhances specificity in predicting reduced pulmonary infection and may bolster the assessment of CF treatment outcomes.

Source: Thorax.

 

Treatment of low bone density in young people with cystic fibrosis: a multicentre, prospective, open-label observational study of calcium and calcifediol followed by a randomised placebo-controlled trial of alendronate.


Background

Long-term complications of cystic fibrosis include osteoporosis and fragility fractures, but few data are available about effective treatment strategies, especially in young patients. We investigated treatment of low bone mineral density in children, adolescents, and young adults with cystic fibrosis.

Methods

We did a multicentre trial in two phases. We enrolled patients aged 5—30 years with cystic fibrosis and low bone mineral density, from ten cystic fibrosis regional centres in Italy. The first phase was an open-label, 12-month observational study of the effect of adequate calcium intake plus calcifediol. The second phase was a 12-month, double-blind, randomised, placebo-controlled, parallel group study of the efficacy and safety of oral alendronate in patients whose bone mineral apparent density had not increased by 5% or more by the end of the observational phase. Patients were randomly assigned to either alendronate or placebo. Both patients and investigators were masked to treatment assignment. We used dual x-ray absorptiometry at baseline and every 6 months thereafter, corrected for body size, to assess lumbar spine bone mineral apparent density. We assessed bone turnover markers and other laboratory parameters every 3—6 months. The primary endpoint was mean increase of lumbar spine bone mineral apparent density, assessed in the intention-to-treat population.

Findings

We screened 540 patients and enrolled 171 (mean age 13·8 years, SD 5·9, range 5—30). In the observational phase, treatment with calcium and calcifediol increased bone mineral apparent density by 5% or more in 43 patients (25%). 128 patients entered the randomised phase. Bone mineral apparent density increased by 16·3% in the alendronate group (n=65) versus 3·1% in the placebo group (n=63; p=0·0010). 19 of 57 young people (33·3%) receiving alendronate attained a normal-for-age bone mineral apparent density Z score. In the observational phase, five patients had moderate episodes of hypercalciuria, which resolved after short interruption of calcifediol treatment. During the randomised phase, one patient taking alendronate had mild fever versus none in the placebo group; treatment groups did not differ significantly for other adverse events.

Interpretation

Correct calcium intake plus calcifediol can improve bone mineral density in some young patients with cystic fibrosis. In those who do not respond to calcium and calcifediol alone, alendronate can safely and effectively increase bone mineral density.

Source: Lancet

 

 

 

Fluid-fluid level in cystic vestibular schwannoma: a predictor of peritumoral adhesion.


Abstract

OBJECT

The aim of this study was to evaluate the clinical results and surgical outcomes of cystic vestibular schwannomas (VSs) with fluid-fluid levels.

METHODS

Forty-five patients with cystic VSs and 86 with solid VSs were enrolled in the study. The patients in the cystic VSs were further divided into those with and without fluid-fluid levels. The clinical and neuroimaging features, intraoperative findings, and surgical outcomes of the 3 groups were retrospectively compared.

RESULTS

Peritumoral adhesion was significantly greater in the fluid-level group (70.8%) than in the nonfluid-level group (28.6%) and the solid group (25.6%; p < 0.0001). Complete removal of the VS occurred significantly less in the fluid-level group (45.8%, 11/24) than in the nonfluid-level group (76.2%, 16/21) and the solid group (75.6%, 65/86; p = 0.015). Postoperative facial nerve function in the fluid-level group was less favorable than in the other 2 groups; good/satisfactory facial nerve function 1 year after surgery was noted in 50.0% cases in the fluid-level group compared with 83.3% cases in the nonfluid-level group (p = 0.038).

CONCLUSIONS

Cystic VSs with fluid-fluid levels more frequently adhered to surrounding neurovascular structures and had a less favorable surgical outcome. A possible mechanism of peritumoral adhesion is intratumoral hemorrhage and consequent inflammatory reactions that lead to destruction of the tumor-nerve barrier. These findings may be useful in predicting surgical outcome and planning surgical strategy preoperatively.

Source: http://thejns.org

Treatment of low bone density in young people with cystic fibrosis: a multicentre, prospective, open-label observational study of calcium and calcifediol followed by a randomised placebo-controlled trial of alendronate.


Background
Long-term complications of cystic fibrosis include osteoporosis and fragility fractures, but few data are available about effective treatment strategies, especially in young patients. We investigated treatment of low bone mineral density in children, adolescents, and young adults with cystic fibrosis.
Methods
We did a multicentre trial in two phases. We enrolled patients aged 5—30 years with cystic fibrosis and low bone mineral density, from ten cystic fibrosis regional centres in Italy. The first phase was an open-label, 12-month observational study of the effect of adequate calcium intake plus calcifediol. The second phase was a 12-month, double-blind, randomised, placebo-controlled, parallel group study of the efficacy and safety of oral alendronate in patients whose bone mineral apparent density had not increased by 5% or more by the end of the observational phase. Patients were randomly assigned to either alendronate or placebo. Both patients and investigators were masked to treatment assignment. We used dual x-ray absorptiometry at baseline and every 6 months thereafter, corrected for body size, to assess lumbar spine bone mineral apparent density. We assessed bone turnover markers and other laboratory parameters every 3—6 months. The primary endpoint was mean increase of lumbar spine bone mineral apparent density, assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01812551.
Findings
We screened 540 patients and enrolled 171 (mean age 13•8 years, SD 5•9, range 5—30). In the observational phase, treatment with calcium and calcifediol increased bone mineral apparent density by 5% or more in 43 patients (25%). 128 patients entered the randomised phase. Bone mineral apparent density increased by 16•3% in the alendronate group (n=65) versus 3•1% in the placebo group (n=63; p=0•0010). 19 of 57 young people (33•3%) receiving alendronate attained a normal-for-age bone mineral apparent density Z score. In the observational phase, five patients had moderate episodes of hypercalciuria, which resolved after short interruption of calcifediol treatment. During the randomised phase, one patient taking alendronate had mild fever versus none in the placebo group; treatment groups did not differ significantly for other adverse events.
Interpretation
Correct calcium intake plus calcifediol can improve bone mineral density in some young patients with cystic fibrosis. In those who do not respond to calcium and calcifediol alone, alendronate can safely and effectively increase bone mineral density.
Source: Lancet

Risk Factors for Bronchiectasis in Children with Cystic Fibrosis.


BACKGROUND

Bronchiectasis develops early in the course of cystic fibrosis, being detectable in infants as young as 10 weeks of age, and is persistent and progressive. We sought to determine risk factors for the onset of bronchiectasis, using data collected by the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) intensive surveillance program.

METHODS

We examined data from 127 consecutive infants who received a diagnosis of cystic fibrosis after newborn screening. Chest computed tomography (CT) and bronchoalveolar lavage (BAL) were performed, while the children were in stable clinical condition, at 3 months and 1, 2, and 3 years of age. Longitudinal data were used to determine risk factors associated with the detection of bronchiectasis from 3 months to 3 years of age.

RESULTS

The point prevalence of bronchiectasis at each visit increased from 29.3% at 3 months of age to 61.5% at 3 years of age. In multivariate analyses, risk factors for bronchiectasis were presentation with meconium ileus (odds ratio, 3.17; 95% confidence interval [CI], 1.51 to 6.66; P=0.002), respiratory symptoms at the time of CT and BAL (odds ratio, 2.27; 95% CI, 1.24 to 4.14; P=0.008), free neutrophil elastase activity in BAL fluid (odds ratio, 3.02; 95% CI, 1.70 to 5.35; P<0.001), and gas trapping on expiratory CT (odds ratio, 2.05; 95% CI, 1.17 to 3.59; P=0.01). Free neutrophil elastase activity in BAL fluid at 3 months of age was associated with persistent bronchiectasis (present on two or more sequential scans), with the odds seven times as high at 12 months of age and four times as high at 3 years of age.

 

CONCLUSIONS

Neutrophil elastase activity in BAL fluid in early life was associated with early bronchiectasis in children with cystic fibrosis. 

Source: NEJM

 

Mannose-binding lectin and innate immunity in bronchiectasis.


Pathogenic microorganisms often thrive in the inflammatory milieu of the bronchiectatic airway where innate and adaptive defence mechanisms can be impaired. Although genetic defects of the adaptive immune system causing immunodeficiency syndromes are well characterised, genetic defects that impair the recognition of microbes by the innate immune system have only recently been identified.1 For example, polymorphisms in the gene for mannose-binding lectin (MBL), a receptor of the innate immune system that recognises microbial carbohydrates, can lead to deficiency of MBL and increased susceptibility to infection.

When the lungs are exposed to a new pathogen, the first line of defence is the innate immune system, which results in a swift and semi-specific response. Cells of the innate immune system, which include dendritic cells and macrophages, recognise highly conserved structures called pathogen-associated molecular patterns (PAMPs) that are shared by large groups of microorganisms. PAMPs are recognised by pattern-recognition receptors, which activate the cells of the innate immune system to rapidly attack and kill microbes.2

MBL is a soluble pattern-recognition receptor that is synthesised in the liver and is released into the systemic circulation as a component of the acute-phase response. It is not produced locally in the lungs and is thought to leak into the airways and alveoli from the systemic circulation, particularly in the presence of inflammation.3 MBL binds to various respiratory pathogens including Haemophilus influenzae and Pseudomonas aeruginosa, which are commonly identified in the airways of patients with bronchiectasis, and enhances the killing of these organisms by activation of the lectin complement pathway and by facilitating phagocytosis by opsonisation.4

In The Lancet Respiratory Medicine, James Chalmers and colleagues report a large, prospective study5 assessing the relation between MBL deficiency and clinical outcomes during a 4 year follow-up of patients with non-cystic fibrosis bronchiectasis. 55 (12%) of 470 patients with bronchiectasis had genotypes associated with MBL deficiency. These patients had more frequent exacerbations and were more likely to be chronically colonised with bacteria, particularly by P aeruginosa, than were patients with genotypes not associated with MBL deficiency. One strength of the study was the measurement of both MBL deficient genotypes and serum concentrations, which were strongly correlated. Serum MBL deficiency (<200 ng/mL) was also associated with increased exacerbation frequency.

The results of Chalmers and colleagues’ study5 are consistent with the findings from studies of patients with cystic fibrosis in which MBL deficiency has been associated with increased severity of disease. In patients with cystic fibrosis, MBL deficiency results in earlier acquisition of P aeruginosa, reduced pulmonary function, and increased mortality.6 However, a retrospective study7 of patients with non-cystic fibrosis bronchiectasis reported no association between low MBL concentrations and exacerbation frequency. Several conflicting results have also been published from studies8 assessing the association between low levels of MBL and acute exacerbations of chronic obstructive pulmonary disease.

What are the clinical implications of Chalmers and colleagues’ study? The study provides evidence that MBL deficiency is a new risk factor for infection and acute exacerbations in patients with non-cystic fibrosis bronchiectasis. Identification of patients at high risk of development of severe disease could direct clinicians to undertake more intensive management and follow-up of these patients with a view to reducing rates of hospital admission and mortality. Such stratification is increasingly relevant because of the growing range of treatments that is emerging for bronchiectasis. These treatments include long-term azithromycin, nebulised gentamicin, inhaled mannitol, inhaled dry powder ciprofloxacin, and nebulised liposomal ciprofloxacin. Recombinant human MBL might also become a treatment option, after it was reported in a phase 1 study9 to be safe, well tolerated, and able to restore activity of the lectin pathway of complement.

A standard definition of clinically significant MBL deficiency is not presently available but a diagnostic approach that incorporates both serum concentrations and genotyping seems sensible. Some patients with genotypes that are not associated with deficiency can still have very low MBL serum concentrations and, alternatively, serum concentrations can increase with the acute-phase response. One pragmatic approach analogous to that recommended by the American Thoracic Society and European Respiratory Society for α1-antitrypsin deficiency might be for clinicians to initially measure the serum concentration of MBL in patients with bronchiectasis. If the serum level is low, genotyping could then be undertaken. A cutoff of 200 ng/mL, as used in the study by Chalmers and colleagues, classified 19% of patients with bronchiectasis as MBL deficient. Better access to testing facilities and further studies are required to confirm the findings of the present study5before testing for MBL deficiency becomes routine practice in non-cystic fibrosis bronchiectasis. The present findings also raise some interesting questions for future research. MBL deficiency is relatively common in the general population and does not seem to predispose to an increased risk of infection in the absence of other predisposing factors. How do other predisposing factors interact with MBL deficiency to increase the risk of infection and cause severe disease or poor longitudinal outcomes in bronchiectasis? Could serial MBL concentrations in serum and sputum be used as markers for early detection of exacerbations or determination of the duration of antibiotic treatment? Does azithromycin interact with MBL to improve phagocytic activity in macrophages? Although some evidence suggests that azithromycin increases mannose receptor (a pattern-recognition receptor in the same family as MBL) expression and phagocytic activity in alveolar macrophages, the effect of azithromycin on MBL expression is unclear.

Source: lancet

bronchie

Study Finds Concerns Around Current Infection Control Practices.


Multidrug-resistant Mycobacterium abscessus is frequently transmitted between patients with cystic fibrosis, suggesting that current infection control is not adequate, according to a Lancet study.

Researchers analyzed 168 isolates from 31 patients with pulmonary M. abscessus infection at a U.K cystic fibrosis center. Within clusters ofM. abscessus subspecies massiliense, isolates from different patients were often more closely related to each other than were isolates from within a single individual, suggesting transmission of the bacteria between patients. In addition, some isolates were resistant to macrolides and aminoglycosides in patients previously unexposed to those antibiotics, supporting the notion of cross-infection. The authors say transmission is likely occurring indirectly, such as through fomite contamination or lung function testing.

The authors conclude that their findings raise “several important questions about current infection control measures used in treatment centers, the potential for cross-infection in other patient groups and with other … species, and whether mandatory notification of infections with M. abscessus complex and routine whole genome sequencing might be required to identify and control the spread.”

Source: Lancet