15 Nucleic acid therapy - Xidianweb.xidian.edu.cn/yqxia/files/20170610_210353.pdf · The first gene therapy 6 Dr. Anderson’s team drew blood from their patient, and replaced the

By Yuqiong Xia6/10/2017

Gene Therapy

Pharmaceutical Biotechnology 西安电子科技大学夏玉琼

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生物技术制药

技术基础

生物技术药物

药物的开发

药物的发现

专利的申请

临床前试验

临床试验

药物的生产生物药物的来源

生物药物的生产和纯化

生物药物的分析

细胞因子干扰素

白介素造血生长因子

生长因子

激素类药物血液制品和治疗性酶

抗体、疫苗和佐剂

核酸药物

生物技术制药概念西安电子科技大学夏玉琼

Introduction to gene therapy

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The use of nucleic acids as therapeutic medicicalcompounds

Compensate for abnormal gene expression

http://genepulse.net/wp-content/uploads/2013/01/gene_therapy_getty.jpg

西安电子科技大学夏玉琼

The first gene therapy

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Ashanti DeSilva was born with a lack of the enzyme adenosine deaminase (ADA).

The root cause is a single defective gene.

Patients with ADA deficiency suffer from severe immunodeficiency.

http://www.lifesciencesfoundation.org/printer_events-The_first_gene_therapy.html


5 http://www.lifesciencesfoundation.org/printer_events-The_first_gene_therapy.html


The first gene therapy

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Dr. Anderson’s team drew blood from their patient, and replaced the defective gene with a functional variant.

The therapy partially restores Ashanti’s immune system. It temporarily stimulates production of the missing enzyme, but does not generate new cells with functional genes.

Ashanti continues to receive injections of corrected T-cells every two months. She is able to lead a normal life.

http://www.lifesciencesfoundation.org/printer_events-The_first_gene_therapy.html


Outline

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In vivo and ex vivo gene therapy Cancer gene therapy Gene delivery vector Gene drug


In vivo vs ex vivo

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In vivo Direct injection of vector/DNA complexes into

bloodstream Intratumoral, subcutaneous, intramuscular injection

Ex vivo Isolation and culture of cellular targets Direct application of the vector for efficient gene

expression


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Outline

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Disease target 西安电子科技大学夏玉琼

Gene therapy for cancer

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Correction of genetic mutations Immunotherapy


Correction of genetic mutations

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Over-expression of tumor suppressor genes such as p53, MDA-7, ARF

p53 is one of the most studied candidates


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The first gene therapy product

重组人p53腺病毒注射液（商品名：今又生）

5型腺病毒载体DNA和人p53肿瘤抑制基因重组


Immunotherapy to treat cancer

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Limitations Limited expression and low avidity(亲和力) of tumor-

associated antigen-specific cytotoxic T-cells Inherent ability of tumor cells to evade immune

detection

Methods Gene transfer to cancer tissue Gene transfer to cancer cells Gene transfer to T-cells


Gene transfer to cancer tissue

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Direct injection of a vector expressing immunostimulatory molecules or tumor-specific antigens in a tumor. As the transgene product is released, macrophages,

dendritic cells, natural killer cells and T-cells are activated and migrate to the tumor where they destroy cells expressing the transgene


Gene transfer to cancer cells

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Cells isolated from the patient or cancerous cell lines are treated with the vector in culture, killed by irradiation and given back to the patient. Epitopes on the cells prompt the immune system to attack

and remove malignant cells


Gene transfer to T-cells

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T-cells or bone marrow cells from the patient are cultured with a vector and/or tumor antigens. Live cells that attack and remove malignant cells are given back to the patient


癌症的免疫疗法的临床应用情况

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第一代 LAK细胞疗法从病人外周血中提取细胞，然后

在体外用IL-2来诱导产生“杀伤性免疫细胞”，最后回输这些细胞到病人体内。

临床无效

第二代 CIK细胞疗法从病人外周血中提取细胞，然后

在体外用IL-2和别的细胞因子来诱导产生“杀伤性免疫细胞”，最后回输这些细胞到病人体内。

临床无效


癌症的免疫疗法

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第三代 CIK-DC细胞疗法同时输入“杀伤性免疫细胞”和

“树突状细胞” 临床无效

第四代 CAR-T疗法提取患者T淋巴细胞，培养改造，

通过载体整合进入到正常T细胞基因序列，形成嵌合抗原受体T细胞（CAR-T），回输CAR-T细胞

临床白血病和淋巴癌有效，有望批准


Outline

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Why gene transfer need a vector?

http://www.nano-lifescience.com/images/dna-transport.gif


The transferred gene

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Gene expression cassette for gene therapy


Vectors for gene transfer

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Virus vectors Retrovirus Adenovirus

Non-viral vectors Electroporation Cationic polymer Cationic liposome


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Structure of Retrovirus逆转录酶病毒西安电子科技大学夏玉琼

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Simple and complex retrovirus genome

LTR: long terminal repeatsgag, pol and env are structural genes


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The retrovirus replication cycle 西安电子科技大学夏玉琼

Advantages of retroviral vector

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Accommodate transgene cassettes of 8 kb Integrate into the host genome “Pseudotyping” Surface glycoproteins are replaced with those from

unrelated virus


Disadvantages of retroviral vector

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Cannot transduce non-dividing cells May disrupt normal cellular processes The virus titer is very low Virus particles are generally unstable Rapidly removed from the systemic circulation


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Adenoviral vectors 西安电子科技大学夏玉琼

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The adenovirus infection cycle 西安电子科技大学夏玉琼

Advantages and disadvantages

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Advantages Biology of the virus is well understood Transgene expression is rapid and robust Physically stable Can infect dividing and non-dividing cells

Disadvantages Elicit a strong immune response


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Electroporation

http://www.btxonline.com/pages/FAQ.html


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Cationic polymers

http://www.thno.org/v04p0240.htm


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Cationic polymers: proton sponge effect

http://www.thno.org/v04p0240.htm

PEI, the most commonly used cationic polymer

1. PEI absorbs proton, and becomes positively charged

2. the osmotic pressure in lysosome increase

3. The lysosome disruptand releasePEI/DNA


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Cationic liposomes

Y. Xia et al. / Biomaterials 79 (2016) 56e68


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Lipoplex-mediated transfection 西安电子科技大学夏玉琼

Viral vectors vs non-viral vectors

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Viral vectors High transduction

efficiency Possible

insertional mutagenesis

Limited cloningcapacity

Non-viral vectors Low insertional

mutagenesis Unlimited cloning

capacity Low transduction

efficiency


Outline

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Gene drug

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Plasmid oligonucleotide


Plasmid

41 https://www.researchgate.net


Introduction to oligonucleotides

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Oligonucleotides (ONs) Short (with or without chemical modification) ribo- or

deoxyribonucleotides

http://www.livescience.com/37247-dna.html

25-mer: 25nt

Duplexes


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The structure of ONs： 1st generation

Advantages: Longer circulation half life 40-60 h

due to binding to serum proteins ( unmodified oligos < 10 min)

Disadvantages:More binding to proteins, being

toxic

硫代磷酸 DNA


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The structure of ONs： 2nd generation

Less toxic than PS-ONs Long circulation half life (30 days) But poor substrate for RNase H, only inhibit

translation by forming a steric block

RNase H cuts RNA in a DNA/RNA duplex


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The structure of ONs： 3rd generation

Superior stability and RNA binding properties Lack RNase H activation → chimeras of DNA/PNA or

DNA/LNA can activate RNase H But poor substrate for RNase H


Antisense RNA

46 http://biowiki.ucdavis.edu/@api/deki/files/46/07_antisense.GIF?revision=1

Antisense RNA


Antisense RNA (asRNA)

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Definition A single-stranded RNA that is complementary to a mRNA

Application: commercial Fomivirsen An antiviral drug, 5‘-GCG TTT GCT CTT CTT CTT GCG-

3‘ (with phosphorothioate linkages) Used in the treatment of cytomegalovirus retinitis (CMV巨细胞

病毒性视网膜炎） in immunocompromised patients, including those with AIDS.

Licensed by the FDA for CMV in Aug 1998. Limitation Lack effective design, biological activity, and efficient route of

administration

http://en.wikipedia.org/wiki/Antisense_RNA


siRNA

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Small interfering RNA (siRNA) is a class of ds RNA, 20-25 base pairs in length

Interfere with the expression of specific genes with complementary nucleotide sequences.

siRNA functions by causing mRNA to be broken down after transcription, resulting in no translation

http://en.wikipedia.org/wiki/SiRNA


siRNA

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siRNA can be produced from dsRNA or enter cells by transfection

http://upload.wikimedia.org/wikipedia/commons/6/6c/RNAi.jpg

20-25 bp

Dicer enzyme

RISC


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siRNA and asRNA

https://image.slidesharecdn.com/lennoxlncrnawebinarns-my-150825133349-lva1-app6891/95/knockdown-of-lncrnas-exploring-rnai-and-antisense-oligo-methods-5-638.jpg?cb=1440518468


miRNA (definition)

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Discovered in 1990s A microRNA (abbreviated miRNA) is a small non-

coding dsRNA molecule (22 nt) found in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression.

http://en.wikipedia.org/wiki/MiRNA


How does miRNA function

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miRNAs function via base-pairing with complementary sequences within mRNA molecules.

As a result, these mRNA strands are silenced.


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How does miRNA function

http://www.nature.com/nbt/journal/v25/n6/fig_tab/nbt0607-631_F2.html

~22 bpmiRNA


Summary

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In vivo and ex vivo gene therapy Differences

Cancer gene therapy Correct gene mutations Cancer immune therapy

Gene delivery vector Retrovirus, adenovirus, electroporation, cationic polymers,

cationic liposomes Gene drug Plasmid, asRNA, siRNA, miRNA


Documents

15 Nucleic acid therapy - Xidianweb.xidian.edu.cn/yqxia/files/20170610_210353.pdf · The first gene therapy 6 Dr. Anderson’s team drew blood from their patient, and replaced the