biotech presentation (gene vectors - tumor targeting)

8/12/2019 Biotech Presentation (Gene Vectors - Tumor Targeting)

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SYSTEMIC TUMOR-SPECIFGENE DELIVERY

Journal of Controlled Release

Spencer Thomas


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TUMOR-SPECIFIC GENE VECTORS: THE MAGIC BUL

Developing a systemic gene delivery system that is tumor specific involves a basic

and then overcoming the natural tropism of that system in order to achieve tumor s

Localized delivery techniques (intratumoral) of therapeutic gene vectors have show

in treating primary tumors with minimal off-site toxicity Undetected or quiescent metastatic cells are unaffectedrelapse

A gene vector delivery system which acts as a magic bullet for cancer cells is on the

Cell-specific transfection or expression could allow

these vehicles to be delivered systemically with

minimal immunogenic or toxicity

Use of biocompatible nanoparticles utilizing active

or passive selectivity can accomplish this

Viral vectors, mesenchymal stem cells (MSCs), and

siRNA delivery systems could accomplish this

Multiplex nanoparticles with all the required functionalities for cancer management may bethe way to realize the magic bullet proposed 100 years ago by Paul Ehrlich to treat cancer


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CHALLENGES OF GENE-VECTORS

Viral system have strong affinity for liver accumulation, whereas n

often results in high expression within the lungs

Systemic delivery requires tumor-specific gene delivery Very few gene targets are specific to tumors or cancer

lineages; target receptors and motifs overexpressed by tumors is more via

Modify therapeutic gene so only expresses in tumor cells that have proper

signature

Insertion of therapeutic gene in on-site and off-site cells can cau

aberrant transcriptional manifestations i.e under-expression/ overof proteins required for normal function

In the case of viral vectors, the vector itself can lead to aberrant cell

alterations

Amount of gene transfection is often too low to have desired/prolo

increasing dose causes off-site toxicity (lung/liver)


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NON-VIRAL & VIRAL VECTORS

Non-Viral

Cationic Lipoplexes

Micellar-like biopolymers

Cationic polymers

Polyethylenimines (PEIs)

Dendrimers

PAMAM (polyamidoamine)

PPI (polypropylenimine)

Viral

Vacinnia virus

Herpes virus

Lentevirus

Adenovirus (not common for gene vectors)


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NON-VIRAL VECTORS

LIPOPLEXES

Composed of cationic lipid and a neutral lipid or

cholesterol, encapsulates (-) charged DNA

Easily interact w/ cell membrane and internalize

through endocytosis

Leads to destabilization of lipid-complex

cytoplasmic delivery of DNA

Low immunogenic potential, and hold larger

payload of DNA

Relatively non-specific delivery toxicity

Tends to accumulate largely in lungs


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LIPOPLEXES (CONT)

PEGylation + conjugation w/ integrin

Highly specific for tumor cells upon systemic admin

Tissue analysis utilizing FISH (luciferase)

Luciferase DNA accumulation two-fold greater in tumor

tissue compared to lung/liver/spleen

130-fold greater accumulation compared to other organs

PEGylation + anisamide

Targets cells overexpressing sigma receptor (cancer)

10-fold greater expression/mg of tumor

When used to deliver IL-2/IL-12 to subcutaneous

neoblastoman tumors (mouse model)

1/3 of tumors eradicated, other 2/3 had markedly

decreased growth


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NON-VIRAL VECTORS (CONT)

Polyethylenimines (PEIs)

Cationic polymers, ability to condense DNA (with large

payload), internalize into cells and facilitate cellular

endosome rupture (proton sponge)

Tends to accumulate in lungs, considerable non-

specific cytotoxicity

Conjugated w/ FGF specific targeting peptide

Increase of luciferase expression was 2-fold greater in

tumor than in lungs (4-fold greater in lungs)

Conjugated to transferrin

Tumor luciferase expression = 100-fold greater than in

lungs

PEGylationimproved delivery to tumor tissue 10-fold;

specificity slightly decreased


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NON-VIRAL VECTORS (CONT)Dendrimers

Used for applications ranging from gene/drug delivery to nano-

engineering, diagnostic sensors

Core + functional tree-like branches outwards w/ high ability to tailorto therapeutic needs

2 commercially available (cationic) dendrimers used for gene delivery

PPI (Polypropylenimine) otherwise known as POPAM

PAMAM (polyamidoamine)

Generally accepted that PAMAM dendrimers accumulate in the lungs

Has previously been demonstrated that therapeutic gene expression could

still be specific to tumor Recent evidence, that PPI dendrimers have innate tumor specificity

when delivered systemically

Accumulation results in tumor-specific gene expression

Lack of research on this particular class of dendrimers

FIGURE: 3rd gen PPI den

(nitrotripropionamide) and


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DENDRIMERS (CONT)

PAMAM

In mouse model, PAMAM dendrimers delivery gathered

primarily in lung but luciferase expression was isolated tothe tumor (see Figure)

PPI

Authors cited 2 studies: Russ et al. & Chisholm et al.

Use of 2nd & 3rd generation PPI dendrimers induced

exogenous gene expression up to 100-fold greater in tumor

compared to all other organ

Conjugated w/ transferrin

4-fold greater tumor tissue expression (compared to

hepatocytes) of gene

TNF-w/ tumor-specific promoter

Complete regression of 90% of tumors (mice model)


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MESENCHYMAL STEM CELLS (MSCSDELIVER SYSTEM

Attractive due to highly proliferative nature, easy

isolation, genetically modified to express genes,

sustained viability and expression (3-21 days)

Observed to have innate ability to accumulate in

tumor tissue magic bullet without extensive

modification/synthesis required

Studies involving MSC integrated w/ IL-12 and IFN-

beta exhibited tumor-specific delivery to gliomas,

melanomas, and breast tumors

Could be artifact of tumor system (mice infected w/

human cancer cell lineage and human derived MSCs)


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VIRAL VECTORS

The utilization of viral techniques to hijack viral vector systems make use of very

mechanisms to internalize their own genome into target cells

Some viruses preferentially infect cancer cells over normal cells

Naturally derived viral vectors can be modified, disabled and used to introduce th

genes into cells

Attractive due to evolutionary adaptations which have proceeded for millions of y

3 main viral vectors for therapeutic gene delivery

Vaccina viru s: possesses gene encoding transcriptional proteins and high gene exp

independent of host cell replication

Herpes virus: preferentially infects cancer cells over normal cells

Lent iv i rus:retroviral, integrate integration of its DNA into cell genome


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VIRAL VECTORS (CONT)Vaccina virus

Puhlman et al. thoroughly characterized systemic vaccina gene deliveryutilized muta

vector (inactivation of TK, luciferase expression)

Systemic IV injection demonstrated luciferase tumor expression to be 250-fold higher than c

affected tissue

Replacing luciferase w/ genes encoded for therapeutic genes successfully treated tumors (ra

(1) Study involving DNA encoded w/ cytosine deanimase demonstrated significant longer life expec

rate

(2) viral particles loaded w/ endothelial activating polypeptide II demonstrated TNF-resistant melaand tumor regression

However, high immunogenic response lead to rapid clearance, nonetheless potential for agg

therapeutic response

Herpes Simplex Virus (HSV)

Study investigating mutated rHSV (replicative ability limited to MEK expressing cells)

Systemic administration (IV) of rHSV vector exhibited tumor-specific expression and significa

growth

Ex vivoimaging of luciferase-independent expression elucidated non-specific distribution

rHSV only activated in MEK expressing (cancer) cells, though non-specific vector delivery achieved


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VIRAL VECTORS (CONT)

Lentivirius

Inherently non-tumor specific

Specificity was found to be increased by: (1) modification of viral envelopeincre

efficiency of gene delivery, (2) mutating E2 envelope protein (3) incorporating P-g

antibody

Study on mice infected w/ melanoma (showing metastatic cells in the lungs)

Ex vivo imaging of Luciferase expression indicated selective to melanoma cells expresglycoprotein in mouse models

More remarkably, metastases in lungs high expression of luciferase whereas, control n

mice had no detectable gene expression w/ limited expression in liver and spleen

Effectively targeted quiescent metastases


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SIRNA DELIVERY SYSTEMS


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CONCLUSION

The efficient and safe delivery of plasmids to target cells upon direct, systemic in

administration remains one of the most important challenges for the development

gene therapy vehicles and eradication of malignant neoplasms

The innate nature of viral vectors and cationic polymers for non-specific tissue ac

especially that of the liver and lungs provides many obstacles.

Harnessing the potential of retroviral vectors would allow the integration of therapeutic

ultimate termination or restitution of normal function in previous cancer-infected cells

There is high hopes for rHSV which has previously shown ligand/antibody targeting m

necessary if gene expression in the organs can be adequately limited using cellular pr

expressed at high levels in cancer cells

PPI dendrimers, even with the limited research conducted on them, show a high l

success demonstrating an interesting ability to act as biological homing mechanis

bullets for tumors.

Systemic delivery and effective transfection of genes, including TNF- and IL-12,

the immunogenic activity of NK cells and superior suicide pathways is a sophistic

target cancer but requires advancement in understanding the molecular biology o

(more so the tumor microenvironment) before any breakthroughs may ensue.


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REFERENCE

Kullberg M, Mccarthy R, Anchordoquy TJ. Systemic tumor-specific gene delive

Release. 2013;172(3):730-6.

biotech presentation (gene vectors - tumor targeting)

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