本期內(nèi)容是 實(shí)驗(yàn)十：病毒載體基因治療，實(shí)驗(yàn)?zāi)M請(qǐng)看下一期。本部分內(nèi)容來(lái)自 University of California, Berkeley - UC Berkeley Extension, 虛擬實(shí)驗(yàn)的內(nèi)容來(lái)自 Labster. 本部分內(nèi)容均不會(huì)標(biāo)記為為原創(chuàng)，但由于是UP主購(gòu)買的課程，因此不接受非授權(quán)的轉(zhuǎn)載，謝謝您的理解。

每一個(gè)生物基礎(chǔ)實(shí)驗(yàn)均會(huì)分為三部分：第一部分為實(shí)驗(yàn)的生物理論；第二部分為實(shí)驗(yàn)的指導(dǎo)手冊(cè)；第三部分為 Labster 的虛擬實(shí)驗(yàn)?zāi)M。第一部分的基本信息由 Ying Liu, Ph.D. 提供，第二部分的實(shí)驗(yàn)手冊(cè)來(lái)自 Labster, 第三部分的實(shí)驗(yàn)?zāi)M過(guò)程由UP主操作。

Virtual Lab Manual 10 -?Viral Gene Therapy

Synopsis

Does using viruses as a form of therapy sound like science fiction to you? This may come as a surprise, but it’s actually a technique on the rise used to deliver functional genes into patients. In the Viral Gene Therapy simulation, you will learn about the use of modified viruses and how to equip them with therapeutic genes. Take advantage of the recent exciting research findings in the field, and try to develop a potential cure for heart failure. Will you be able to design a virus that can help to revert heart failure in patients?

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Using viruses to cure heart failure

The student will attend a seminar highlighting the causes and symptoms of heart failure, and will have to use gene therapy as a promising way for a treatment based on a particular gene. The student will learn the principles behind the concept of gene therapy and pseudovirus production, and apply them to the present study case of heart failure.

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Pseudovirus production and testing

The student will perform a fitness test with mice to validate a gene as a therapeutic target for gene therapy. Next, they will have to produce the viral capsids containing the therapeutic gene through transfection of the relevant plasmids and observe them through an electron microscope. Finally, the efficiency of the produced viruses will be tested on mice again. Since this is a virtual lab, no real animals will be harmed in this experiment, and results are available immediately compared to the expected 5 weeks of incubation.

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Test your gene therapy treatment

Once you have designed your gene therapy treatment and produced your viruses containing a therapeutic gene, it will be time to test it on mice affected with heart failure. Will you be able to find a cure for this genetic condition?

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Learning Objectives

At the end of this simulation, you will be able to…

●?Explain the use of gene therapy for the treatment of heart failure

●?Explain the causes of heart failure

●?Design a viral mediated?gene therapy approach

●?Define “therapeutic gene”

●?Describe the anatomy and function of the heart from a healthy person vs. a heart failure patient

●?Produce replication defective?recombinant adeno associated?virus (rAAV)

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Techniques in Lab

●?Co-transfection of mammalian cells

●?Viral vector production

●?Electron microscopy

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Theory

Gene Therapy

Gene therapy is an experimental technique for treating disease by altering the patient's genetic material. Most often, gene therapy works by introducing a healthy copy of a defective gene (also called?therapeutic gene) into the patient's cells. Different vectors exist that carry and deliver the therapeutic gene into the patient's cells.?Viral vectors?are most commonly used, but also non-viral methods exist, such as injection of naked?DNA?or DNA complexes.

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Adeno-Associated Virus (AAV)

The Adeno-associated virus (AAV) is a small, single-stranded DNA virus that belongs to the family of?Parvoviridae.

AAV infects humans and other primates; however, it is not known to cause any disease. The lack of pathogenicity makes AAV attractive for its application as a?viral vector?for?gene therapy.

AAV structure and genome

AAV is characterized by an icosahedral capsid and a genome size of 4.7 kb. The genome contains two open reading frames, encoding for?rep?and?cap. Rep is composed of four genes encoding for viral proteins required for replication. Cap contains VP1, VP2, and VP3, which together build the virus capsid.

AAV in gene therapy

The desired?therapeutic gene?cDNA is inserted between two inverted terminal repeats (ITRs) that aid the packaging of the viral genome into the virus capsid.

Once infected with a recombinant AAV, the introduced therapeutic gene will not integrate into the host genome but remain as episomal concatemers in the cell nucleus. Consequently, AAV DNA is lost upon cell division as the episomal DNA is not replicated.

Compared to other viral vectors, AAV has a rather small cloning capacity and requires the complete replacement of the 4.7 kb genome. This allows for the incorporation of only small therapeutic genes.

The application of AAV as a viral vector for gene therapy is limited by the fact that some patients were unnoticedly infected with AAV and therefore produced neutralizing antibodies against the virus, which impairs the efficiency of the treatment.

Cardiac Cycle

Cardiac cycle is the period of time that begins with contraction of the atria and ends with ventricular relaxation. The cardiac cycle includes all events associated with the blood flow through the heart during one complete heartbeat.

The cardiac cycle begins with atrial systole and continues?to ventricular systole, atrial diastole, and ventricular diastole.

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Heart Failure

Heart failure, sometimes also called congestive heart failure, describes a condition in which the heart is not able to pump sufficient blood to cover the body's needs. In particular, the heart wall, or myocardium, is dilated in heart failure patients.

Heart failure is a fatal condition and the most common reason for hospitalization of people above the age of 65.

The causes of heart failure include coronary artery disease, high blood pressure, heart attacks, or conditions that overwork the heart.

Typical symptoms of heart failure are shortness of breath, fluid retention (e.g. swollen ankles, legs, abdomen), reduced ability to exercise, weakness and fatigue, rapid or irregular heartbeats. Heart failure can be triggered for example during physical exercise when the heart is under strenuous effort.

Conventional treatments include ACE-inhibitors, beta-blocker, diuretic, and Angiotensin II Receptor Blockers (ARB's). However, most of the commonly available drugs have high side effects, and many patients still have a poor life expectancy despite treatment.

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Recombinant adeno-associated?virus (AAV) production

Several systems exist to produce recombinant adeno-associated virus (AAV). The most common system is based on a 3 plasmid co-transfection (Figure 2) of HEK293 cells as the packaging cell line. Following?lipid based?co-transfection, cells are harvested by lysis, and the virus is purified for infection of target cells (Figure 3).

Use of animal models in research

Animal models are used in research in order to perform experiments that would otherwise not be possible; for instance, due to time restrictions when studying development, or because of ethical reasons when studying human diseases. The results are then extrapolated to human beings.

The criterion used to select the animal model depends on the objective of the study, but one of the most important considerations is the degree of conservation of the studied process in humans. Thus the similarity of the animal model is very important when aiming to apply the conclusions of the experiment to humans. The most common animal models utilized in research are mice and rats.

The main disadvantage of this approach is the need of maintaining expensive animal facilities. Usually, research centers have a common service that is shared by all research groups.

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Transmission Electron Microscopy

Transmission Electron Microscopes (TEM) work similarly to light microscopes, but they utilize electrons instead of light. The electrons pass through the sample like light through a shadow puppet screen. Dense structures absorb a lot of electrons and create a dark spot on the resulting image, just like the shadow of a puppet blocking the light. A TEM image is always black and white; staining techniques only allow increasing the density of certain structures and thereby making them appear darker. To bundle the electrons, the TEM contains strong magnets that are analogous to lenses in the light microscope. To efficiently illuminate the specimen with an electron beam, the sample slice needs to be very thin, and the body of the TEM has to be evacuated.

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