L17 - Gene Therapies I

Intro Gene Therapy

A technique that allows doctors to treat a disorder by modifying a gene into a patients cells instead of using drugs or surgery

• new gene into body to help fight a disease

• inactivating mutated/malfunctioning genes

• replacing a mutated gene with a healthy copy

Applications

• defective CFTR gene replacement for cystic fibrosis

• supplying large quantity of factor IX in hemophilia

• introducing genes into a cell that do not normally express it such as “immunopotentiation genes” or “suicide genes” in cancer cells

• introducing protective genes into target cells such as viral-specific ribozymes to treat HIV-1 infections

• modulation of immune response such as tolerance induction in autoimmune diseases

• DNA vaccination such as West Nile virus for veterinary applications

Requirements for Success

• selecting the right gene

• iding and accessing target cells for treatment

• appropriate gene delivery system

• proof of principle, safety, and efficacy

• suitable manufacturing and analytical processes

Target Cells

Somatic Gene Therapy

• noninheritable

• only expressed in target cells

• aimed to cure only the patient not patient’s descendents

• the only gene therapy that has been applied in humans

Germline Gene Therapy

• inheritable

• genetic modification will pass the selected changes to the next gen

• widely used in experimental animals in the form of transgenic or “knockout animals in which exogenous genes is either introduced or deleted respectively

Advantaged of Targeting Lymphocytes for Somatic Cell Gene Therapy

• relatively long lived

• readily obtainable from peripheral blood

• easy to manipulate

• no inactivation of gene expression during differentiation

• can be depleted post-transfer as a safety backup

• can be used to supply blood borne gene products; able to secrete large amt of protein (hemophilia B - factor IX)

• potential in manipulating immune response - Car-T cell immunotherapy

Ex Vivo vs In Vivo

Ex Vivo - host cells are taken and genetically modified and amplified containing therapeutic gene and injected back into patient

• Advantages:

  • does not require tissue specific vectors

  • very high transfer efficency

  • target cells can be manipulated/amplified

• Cons:

  • can be used only for limited target cells such as blood cells

  • cells need to retain the ability to “home” and function normally post transfer

  • In vitro artifacts

In Vivo - injectable vectors is administered to the patient

• Advantages:

  • can target all body tissue

  • No in vitro artifacts

• Cons:

  • specificity of gene transfer could be an issue

  • less invasive

Gene Delivery Systems

Harmless Viruses - viral vector

• virus genes are removed and replaced with the gene of interest

• packaged into viral particles to smuggle genes into target cells by infecting them

• some can not only carry genes into the cell but also integrate the gene into host cell chromosomes

Retroviruses - Single-stranded positive sense RNA virus

• inserts a copy of its genome into DNA of host cell changing the cell’s genome

• infection will persist indefinitely

• Right RNA packaged into the virion particle

  • RNA → dsDNA → RNA → polypeptide

• Cis sequences - directly active as nucleic acids

  • 5’ long terminal repeat (LTR) - a transcriptional promoter that contains sequences important for the reverse transcription of the genome in RNA form

  • primer binding site (PBS) - first strand DNA synthesis during reverse transcription

  • psi sequence - directs packaging of the genomic RNA into the virion

  • polypurine tract (PPT) - primer binding site for 2nd strand DNA synthesis during revers. trans.

  • 3’ LTR - acts a s polyadenylation signal in dna form, contains sequences for revers. trans. in RNA form

• Trans - protein coding sequence

Replication defective retroviral vector system - separating the cis and trans genetic functions

• vector construct - contains cis seq.

• helper/packing plasmids - encode viral proteins

Simplest Retroviral Vector - trans seq. replaced w/ gene of choice is limited to expressing only one gene

Strategies to express multiple genes from one Viral Vector

• expression of diff prieins form alternatiely spliced messenger RNAs transcribed from one promoter

• use of IRES elements allow translation of multiple coding regions from a single mRNA

• use of the promoter in LTR and internal promoters to drive transcription of diff cDNAs

Advantages of Retroviral Vectors:

• efficient and stable integration

• controllable host range via envelope psuedotyping

• capable of delivering up to 8kbps of exogenous sequences

Cons:

• can only infect dividing cells

• difficult to obtain high titers (high conc.)

• 8kbps may not be enough

Safety Concerns

• production of replication-competent virus - separating gag-pol and env genes such that assembly of the entire viral genome requires 2 non-homologous recombination events (low poss.)

• Insertional Mutagenesis - integration can activate a protooncogene (reported only in IL2RG gene therapy thus far)

Lentiviral vectors - more complex type of retroviruses

• ability to infect both dividing + non cells

• integrate permanently into host genome

• human lentiviruses that have been explored for gene delivery systems are HIV-1 and HIV-2

Adenoviral Vector Delivery System - non enveloped double-stranded DNA virus

• Delivery System

  • binds to receptors on surface and enters via endocytosis

  • escapes endosomes and travels to nucleus

  • DNA enters nucleus (not integrate into genome)

  • host machinary transcribes delivered gene

  • protein produced

• can be replication defective

• high titers!

• can infect broad range of dividing + non cells

• strongly immunogenic

• limitations of first gen

  • pre-existing immunity

  • leaky expression from genes that were not deleted

  • limited # of repeat treatment

• improved vector

  • high capacity - multiple genes can be transduced in one shot

    extended time of gene expression

  • reduced immunogenicity

• Main advantages:

  • no evidence for chromosomal integration

  • viral genome stable and does not undergo rearrangement at high rate

  • low pathogenicity of virus in humans

  • and see above^^

• Main disadvantages:

  • very immunogenic

  • do not integrate into host genome

  • transient expression

• could be good for:

  • modulating immune response

  • developing vaccines

  • cancer treatments

Adeno-Associated Viral (AAV) Vectors - non-enveloped single stranded DNA virus

• non-pathogenic, defective viruse that requires helper virus to supply machinery for producing infectious particles

• standard AAV vectors package single-stranded DNA after infection:

  • virus enters cell and delivers ssDNA into nucleus

  • host must convert ssDNA → dsDNA before transc.

  • second strand synthesis is often slow and inefficient esp. in non-dividing cells

• Designing new AAV vectors

  • having temporally modulatable expression

  • use a muscle-specific promoter to drive gene expression

  • can target viral vector to specific cells using designer cap protein

  • can use bidirectional promoters to drive mult. gene expression

• Main Advantages:

  • small, easy manipulation

  • infect various dividing + non

  • low immunogenicity

  • not associated w/ known human diseases

• Main Disadvantages:

  • limited packaging capacity (~5kb)

  • requires adenovirus as helper

Other Viral delivery systems

• Herpes Simplex Type I Virus (HSV) - double stranded dna

  • neurotropic (tissue specific gene transduction)

  • treat cns disease ex. parkinsons

  • can be selectively depleted by treating with Ganciclovir (naturally encodes HSV thymidine kinase HSV-TK gene