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Gene Therapy

Pol Huedo Moreno Genómica y Proteómica

Junio 2010

Definition

“The controlled introduction of therapeutic nucleic acids into target cells to complement genetic deficiencies, or short nucleic acids (either antisense or interfering-based RNAs) to block the expression of specific genes associated to particular cell malfunctioning or parasite multiplication”.

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What is gene therapy?

Imagine that you accidentally broke one of your neighbor's windows. What would you do? You could:

1. Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed. 2. Try to repair the cracked window with some tape: not the best long-term solution. 3. Put in a new window: not only do you solve the problem, but also you do the honorable thing.

So, if a flawed gene caused our "broken window," can you "fix" it? What are your options?

1. Stay silent: ignore the genetic disorder and nothing gets fixed. 2. Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution. 3. Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!

What is gene therapy?

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-  Cancer (mainly in lung, breast, prostate and colon)

-  Cardiovascular -  Monogenic disease -  Infectious -  Neurological diseases

Targets of Gene Therapy

Types of gene therapy

Germ line gene therapy

- Sperm or eggs modified by the introduction of functional genes. - Heritable. - Highly effective in counteracting genetic disorders and hereditary diseases. - Many jurisdictions prohibit this for application in human, for a variety of technical and ethical reasons.

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Somatic gene therapy

- Somatic cells. - Non heritable. - Lower efficience - Jurisdictions allow this aplications. - Typical gene therapy.

Types of gene therapy

Ex vivo gene therapy

Surgically removing cells from the affected tissue area, injecting or splicing the new DNA. The new tissues are placed back into the affected area of the

patient. Ex: Leukemia.

Types of gene therapy

www.biochem.arizona.edu/.../AMG9.11b.gif

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In vivo gene therapy

No surgery or even anesthesia. In this process, the therapeutic DNA is transferred directly into the body cells.

Types of gene therapy

www.biochem.arizona.edu/.../AMG9.11b.gif

Strategies Toward Gene Therapy

The AAPS Journal 2006; 8 (4) Article 83 (http://www.aapsj.org).

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Viral Vectors

Non Viral Vectors

Gene therapy vectors

Viral Vectors Adenovirus Retrovirus Lentivirus Adeno-associated virus Herpes Virus (…)

Gene therapy vectors

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Non Viral Vectors - Naked DNA - VLPs

Gene therapy vectors

·Polyplex ·Lipoplex ·Artificial virus

The AAPS Journal 2006; 8 (4) Article 83 (http://www.aapsj.org).

Currently Used Vectors

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Clinical Trials 2007

Currently Used Vectors

Gene therapy vectors Viral Vectors

-  In vivo/Ex vivo

- High Efficiency

- Immune response

- Inflammation

- Genome alterations (mutations)

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Viral Propieties that are relevant to gene therapy.

We have to imitate this propieties

Gene therapy vectors Viral Vectors

Gene therapy vectors Viral Vectors

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Gene therapy vectors Viral Vectors

Recognize

Endocitocis

Uncoating

“Integration”

Expression

Stability

Gene therapy vectors Viral Vectors

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Gene therapy vectors Non Viral Vectors

-  In vivo/Ex vivo

- Mild Immune response

-  No Inflammation

- Low Efficiency

- Genome alterations (mutations)

Gene therapy vectors Non Viral Vectors

Non Viral Vectors - Naked DNA - VLPs -Polyplex -Lipoplex

-Artificial Virus

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Gene therapy vectors Non Viral Vectors

1. Naked DNA

- Safer - Easy, fast, cheap - Mild I.R. - Few minuts on blood - Low efficiency - Bad delivery

Gene therapy vectors Non Viral Vectors

1. Naked DNA

- Electroporation (muscle)

- Au bombing

- Calcium Phosphate

- DEAE-Dextran

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“Virus Like Particles”

Gene therapy vectors Non Viral Vectors

2. Polyplexes (pEI, pLL)

Mastrobattista et al. Nature Reviews Drug Discovery 5, 115–121

Gene therapy vectors Non Viral Vectors

pLL: poly (l-lysine) pEI: poly(ethylenimine)

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2. Polyplex

Nanoaparticle Complex formation through charge-charge interaction between DNA

Pysiological deshielded at low pH

Gene therapy vectors Non Viral Vectors

2. Polyplex

Gene therapy vectors Non Viral Vectors

Molecular conjugates

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2. Polyplex: bottleneck

J. R. Soc. Interface (2010) 7, S67–S82 doi:10.1098/rsif.2009.0260

Gene therapy vectors Non Viral Vectors

3. Lipoplexes (DOTMA, DOTAP).

Mastrobattista et al. Nature Reviews Drug Discovery 5, 115–121

Gene therapy vectors Non Viral Vectors

DOTMA: N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium choloride

DOTAP: 1,2-dioleoyl-3-trimethylammonium propane

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3. Lipoplexes

Gene therapy vectors Non Viral Vectors

3. Lipoplexes

Gene therapy vectors Non Viral Vectors

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Gene therapy vectors Non Viral Vectors

4. Artificial Virus

Mastrobattista et al. Nature Reviews Drug Discovery 5, 115–121

Gene therapy vectors Non Viral Vectors

Mastrobattista et al. Nature Reviews Drug Discovery 5, 115–121

4. Artificial Virus

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Applications

Gene therapy clinical trials, 2007.

Applications

Phases of gene therapy clinical trials, 2007.

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Mastrobattista et al. Nature Reviews Drug Discovery 5, 115–121 (2006)

Applications

Applications

Cancer

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Conclusions

· Short-lived nature of gene therapy. · The biology of human gene therapy remains complex and many

techniques need further development. · Many diseases and their strict genetic link need to be understood

more fully before gene therapy can be used appropriately. · The public policy debate surrounding the possible use of

genetically engineered material in human subjects has been equally complex.

· Not as promising as expected, by the moment. · However, it will represent a very important field in the future

biomedicine.

Artificial viruses: a nanotechnological approach to gene delivery. Enrico Mastrobattista, Marieke A. E. M. van der Aa, Wim E. Hennink and Daan J. A. Crommelin. NATURE REVIEWS | DRUG DISCOVERY, 2006.

Synthetic biology: applications come of age. Ahmad S. Khalil* and James J. Collins. NATURE REVIEWS Genetics, 2010.

Optimizing Targeted Gene Delivery: Chemical Modifi cation of Viral Vectors and Synthesis of Artifi cial Virus Vector Systems. Sabine Boeckle and Ernst Wagner, The AAPS Journal, 2006.

Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection. Francis C. Szoka Jr, et al., Journal of Controlled Release, 2009.

Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery. Christopher L. Grigsby and Kam W. Leong. J. R. Soc. Interface, 2010.

Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. MARK A. KAY, JOSEPH C GLORIOSO & LUIGI NALDINI. NATURE MEDICINE, 2001

Synthetic polymeric vectors in gene therapy. Patit P. Kundu, Vinay Sharma. Current Opinion in Solid State and Materials Science 12, 2008.

Adeno-Associated Virus Vectors in Clinical Trials. BARRIE J. CARTER, HUMAN GENE THERAPY, 2005.

Progress and prospects: nuclear import of nonviral Vectors. AP Lam and DA Dean. Gene Therapy (2010).

References

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THE END