Defects in Ion Channels as a Cause of Inherited Disease ----Cystic Fibrosis

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Glossary Cystic fibrosis (CF) 囊

性纤维化 CFTR (cystic fibrosis tra

nsmembrane conductance regulator) 囊性纤维化跨膜电导调节因子

ABC(ATP-binding cassette) transporter superfamily ABC 转运蛋白家族

cAMP-regulated chloride channel 环腺苷酸氯离子通道

epithelial cell 上皮细胞

Adenovirus (AD virus)

腺病毒 Liposome 脂质体

Cystic Fibrosis

Background & Mechanism

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Genetic defects & Therapy Perspective

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Defects in ion channels caused by gene mutations may lead to many diseases, because these disorders affect the movement of ions across the plasma membrane of excitable cells, thus reduce the ability of these cells to develop or transmit impulses.

A brief introduction

Cystic fibrosis (CF) is the best-studied and most common disease of this kind, which occurs on the epithelial cells.

CF affects various organs, including the intestine, pancreas, sweat glands, and reproductive tract, but it affects the respiratory tract most severely.

Symptom

Victims of CF produce a thickened, sticky mucus( 粘液 ) that is very hard to propel( 推 ) out of the airways( 呼吸道 ). The patients often suffer from chronic( 慢性的 ) lung infections and inflammation, which progressively destroy the lung’s function.

On average ,1 out of 25 persons of Northern European carry one copy of the mutant gene. So about 1 out of every 2500 of them(1/25×1/25×1/4) is homozygous( 纯合的 ) at this locus( 位点 ) and born with CF.

The gene

Isolated by 徐立之 (Lap-Chee Tsui) in 1989 。

-----"Knowing science can enrich your life. Basically, science is a foundation for genuine common sense."

Once both of the sequences of the gene and the amino acid was known, it turns out that the polypeptide was a member of the ABC transporter superfamily.

The protein was named CFTR.

ABC transporter

They are ubiquitous( 无处不在 ) in biology and power the translocation of substrates across the membrane, both in and out, often against a concentration gradient, by hydrolyzing( 水解 ) ATP .

The protein was purified and incorporated into artificial lipid bilayers, it acts as a cAMP-regulated chloride (Clˉ) channel rather than a transporter and also functions in conductance of bicarbonate ions(HCO3ˉ).

It has 12 transmembrane spanning domains, two nucleotide binding domains, and a regulatory domain in the center. 

Two explanations

1. CFTR deficiency Clˉ efflux

volume of fluid

viscosity( 粘性 ) of the mucus

The function of the cilia to push bacteria out

2 ． p.aeruginosa （绿脓杆菌） belongs to the pseudomonas( 假单胞菌属 ). It can bind to the extracellular end of the CFTR protein, which leads to the ingestion and destruction of the bacterium by the epithelial cells. Patients who lack the CFTR can’t clear the bacterium out.

It is also speculated( 推测 ) that the decreased HCO3ˉsecretion could lower the pH of the fluid in the airway and provide a more hospitable environment for the bacterium.

The deficiency of the CFTR may also leads to the sterility( 不孕不育 ). Because the decrease of the HCO3ˉsecretion affects the vigor of the sperms.

But why did the gene reach such a high frequency in the population instead of dying out?

So heterozygotes( 杂合体 ) may possess some selective advantages over those lacking a copy of the defective gene.

One proposal is that the heterozygotes is protected from the effects of cholera( 霍乱 ).

And the other is from typhoid( 伤寒 ).

to be continued…

Genetic defects & Therapy Perspective

Genetic defects of CF Gene Therapy researched at present Problems and perspective

Genetic defects

By now ,over 800 differernt mutations have been found connected with CF

But almost 70% of the alterated alleles（等位基因） contains the same kind of genetic mutation--△F508

So, what is the △F508 indeed?

!!!Figure 2

The △F508 is a kind of deletion that occurs in the 10th exon of CFTR gene

The missing section are 3 base pairs which encode a pennylalanine （苯丙氨酸） at position 508

With this defection ,the CFTR polypeptide can’t move normally from the endoplasmic reticulum to the surface of epithelial cell（上皮细胞）

So the number of transponters

decreases ,which cause the problem

mentioned above

Gene Therapy researched at present

The major goal of gene therapy is to replace the defective gene with a normal version.And we can make it in vitro（体外） or in vivo （体内） .

The critical problem to solute is: What type of delivery system we choose to conduct the normal gene.

Two delivery systems we used today:

Adenovirus （腺病毒）

Liposomes （脂质体）

Adenovirus

a kind of

icosahedral

virus （二十 面体病毒）

Adenovirus

In 1994,American scientists used the recombinant adenovirus to infect the cells of the airway and deliver the normal CFTR gene successfully.

Advantage:

High efficiency to aim at the target cells

Liposomes

Liposomes

If a small amount of phosphatidylcholine（卵磷脂） is dispersed in an aqueous solution ， the phospholipid （磷脂） molecules assemble to form the walls of fluid-filled spherical vesicles.

Functions: It can be used as vehicles to deliver drugs

or DNA molecules.The drugs or DNA molecules are linked to the lipid bilayer wall or contained at high concentration within its lumen （内腔） .

Liposomes

We use the positively charged liposomes to deliver the normal DNA into the cytoplasm.

Advantage:

Less likely to stimulate a destructive immune response

Problems and perspective

As to adenovirus:

The stability of DNA

The randomicity of the integration （整合） Over transcription of the gene of virus

Distructive immune response As to liposomes:

Less effective in achieveing target cells

Easy to be phagocytized （吞噬）

Perspective

To obey the rule of security and efficiency ,we try to combine the virus and liposome carriers:

e.g. The AD virus-liposome (its efficiency of expression reach up to 30%~40% in the epitheliums （上皮细胞）

Other methods ：

Gene suture （基因缝线） Gene needle Gene carrier combined with solubility fibrin

ogen （可溶性纤维蛋白原）

To solve the problem of the intracellular decrease of the normal DNA, we can first wipe off the virose gene of AD virus and then link it to the plasmids by covalent bond（共价键） .Thus we produce a new kind of carrier.

To improve the targeted transfer （定向转运）， we can combine certain antibodies or peculiar components to the carrier.

TO date ， there is no significant clinical trials of gene therapy on such kind of disease like CF.

Further research are in urgent need!

CCDDY.