Pattern of inheritance for Hemophilia B (X-linked recessive)(1)

Hemophilia B is a X-linked recessive inherited coagulaton disorder that is resultant of a defect in the gene encoding coagulation factor IX, an essential component of the blood coagulation pathway. It is well known as the disease carried by Queen Victoria which affected most of the royal houses of Europe. The concentration of factor IX in individuals with severe hemphilia is less than 1% than that in unaffected inidivuals.

People affected by hemophilia B suffer from bleeding episodes which are associated with arthropathy (bleeding within the joints), internal bleeding from minor injuries, and early death. Previous treatments include frequent IV injection of factor IX protein concentrate, but this treatment does not cure the disease and is very expensive costing $300,000 annually and around $20,000,000 over the course of a lifetime. 

Gene Insertion into AAV VirusesEdit

AAV genomes are ssDNA and are comprised of inverted terminal repeats (ITRs) at each end and two open r


Producing AAV Vectors

eading frames (ORFs) rep (four overlapping  genes encoding life cycle proteins) and cap (overlapping nt sequences of  capsid proteins VP1, VP2, and VP3) (6). In terms of usage for gene therapy, the ITRs are the only elements required in cis next to a thernjknkjnapeutic gene and cap and rep genes can be delivered in trans. Because of these factors, there are multiple methods for production of recombinant AAV vectors containing therapeutic genes.

Serotype 2 (AAV2) StructureEdit

AAV2 presents natural tropism towards skeletal muscles, neurons, VSMCs, and hepatocytes (6). 3 receptors have been illustrated for AAV2:

1. Heparan sulfate proteoglycan (HSPG): Primary receptor

2. avb5 integrin: co-receptor activity enabling AAV to enter the cell by receptor-mediated endocytosis

3. Fibroblast growth factor receptor 1 (FGFR-1): co-receptor activity 

AAV genome organization

AAV genome organization (7)

enabling AAV to enter the cell by receptor-mediated endocytosis There are 2 promoters, p5 and p19, on the left side of the AAV genome, from which two mRNAs that are overlapping, but different lengths can be produced. Each contains an intron that may or may not be spliced. Because of this, 4 various mRNAs and thus 4 different Rep proteins ("left" side) with overlapping sequences can besynthesized. On the other side, the "right" side, there are 3 overlapping sequences encoding capsid proteins VP1, VP2, and VP3, which start from a common promoter, p40. All three are translated from one mRNA, which can subsequently be spliced in two different ways depending on if the longer or shorter intron is spliced, resulting in the formation of two pools of mRNAs of different lengths. 

Generation of recombinant AAV vectorsEdit

A plasmid containing the terminal repeates and the replication (rep) and capsid (cap) genes of AAV2 serotype have been most commonly used. The rep-cap fragment can be replaced by the gene cassette of interest, because as stated earlier, only the terminal repeats are needed for packaging. A protocol using transient transfection is utilized to get the gene cassette of interest into the AAV genomes. However, AAV serotype 2 has been shown to have only transient expression due potentially to the immune response it illicits. 

AAV Vector-Mediated Gene Transfer to Treat Hemophilia BEdit

Aav r vector

Self-Complementary AAV Vector (8)

To get around the immune response illicited by AAV2, researchers at St. Jude are investigating a novel approach towards targeting the vector that is unique to previous clinical trials in 3 ways. First, the factor IX cassette that they developed was packaged as complementary dimers within a single viral particle because it has been shown that self-complementary AAV vectors mediate transgene at higher levels than ssAAV vectors. Second, to get around the antibody-mediated immune response, they pseudotyped the vectors with capsid of serotype 8 (AAV8) because AAV8 is less common than AAV2 and thus is less likely to
Scaav very promising in mice

Self-Complementary AAV Vectors had substantial correction of clotting in mice

illicit an immune response. Finally, AAV8 preferentially targets liver cells, the production site for factor IX. This preferential targeting of the viral vector allows the AAV8 viral vectors to be injected directly into a peripheral vein, which is both non-invasive and very effective because once circulation brings the viral particles to the liver, they infect the hepatocytes and thus transfers the genetic information encoding factor IX (8). 
Factor ix

Role of Factor IX in the Coagulation Pathway (2)

Gene Therapy for Hemophilia BEdit

A delivery system using an adeno-associated virus (AAV) carrying a good version of the human gene for factor IX was used by Dr. Nathwani at the University College London in England to undergo somatic cell gene therapy to treat Hemophilia B (3). The virus is an adeno-associated virus-8, and was an attractive option for researchers because this particular virus generally stays outside the chromosomes, so it did not have the same problem that many gene therapies using viral deliveries have with random insertion of viral DNA into chromosomes, with the ability to disrupt chromosomal genes. Another factor that makes the adeno-associated virus-8 a good option for gene therapy delivery is that patients have little to no immune response to the virus, which infects rhesus monkeys. Finally, this particular virus is attractive because it affects both dividing and non-dividing cells and has long-term, non-toxic expression. A downfall to this virus in general, but not in this case, is that it is small so it only has the ability to carry small genes. In this case, AAV is attractive because the gene encoding factor IX is small enough to be incorporated in AAV.

This particular virus targets liver cells, which is beneficial because the liver cells are the natural site of factor IX production. A potential downfall to this target cell type though is that liver cells do not live forever, so there is some question as to how long the therapy will last due to the slow replenishment of liver cells. Another side-effect with lower risk is the chance that patients will develop liver cancer, which has been observed in mice, so trial participants are monitored closely for that possibility as well.

Aav delivery of therapeutic genes to target tissues

AAV Delivery of Therapeutic Genes to Target Tissues (4)

 A subsequent problem with this approach was that the  affect of the treatment was found to be only transiet (not permanent treatment) because the capsid proteins coating the virus stimulated an immune response destroying host cells containing the new virally-inserted DNA. Dr. Nathwani states that next steps will be to optimize the dosage of the virus so that is is virulent enough to insert its DNA into host cells, but not so virulent that it will trigger an immune response, removing any host cells that have taken up the viral DNA before they have even produced any functional factor IX. 20 more patients will be treated to assess the dosage of the virus to find the highest dosage that does not illicit an immune response (3). One study in dogs and mice found that incorporation of proteasome inhibitors along with the therapeutic gene enhanced gene delivery (5), so that is another way to inhibit immune responses from the host.


1. Inheritance of Hemophilia (photo):

2. Role of Factor IX in Coagulation Pathway (photo):

3. Wade, Nicholas (2011). NY Times: "Treatment for Blood Disease is Gene Therapy Landmark" on research done by Dr. Nathwani (University College London, England):

4. AAV delivery of therapeutic genes into target tissues (photo):

5. Monahan, PE The American Society of Gene & Cell Therapy (2010). "Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application":

6. Adeno-associated virus (Wikipedia):

7. AAV genome organization (photo):

8. St. Jude Children's Research Hospital: Novel Gene Therapy Approach for Hemophilia B: Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O'Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med Dec 22;365(25):2357-65, 2011. Epub 2011 Dec 10. PubMed PMID: 22149959; PubMed Central PMCID: PMC3265081