Teràpia  Gènica  Retroviral  

Cris4na  Fillat  Grup  de  Teràpia  Gènica  i  Càncer  

IDIBAPS    

Família  Retroviridae    

 Virus  4po  C        V.  Leucemia  del  ratón  (MuLV)  

           V.  Sarcoma  murino  (MSV)  Virus  Bpo  B    Virus  Bpo  D    Virus  similares  a  leucosis  aviar    HTLV-­‐BLV    Len4virus          V.  de  la  inmunodeficiencia  humana  Bpos  1  y  2  (VIH-­‐1,  y  VIH-­‐2)    

         V.  de  la  inmunodeficiencia  felina  (FIV)            V.  de  la  inmunodeficiencia  de  simio  (SIV)    

 Espumavirus  

Géneros          Especies  usadas  como  vectores  en  terapia  génica    

I. Overexpress a particular gene in cells difficult to transfect

II. Down-regulate or knock-down a gene to study its function in a particular pathway

III. Gene Therapy

Retroviral    vectors  in  Biology  and  Medicine  

Virus  4po  C.  Oncoretrovirus  

Virions 80-100 nm RNA linear, ss, 7-12 kb

Gag (group antigens): matrix, core proteins Pol: reverse transcriptase: polymerase,RNaseH; integrase Env: envelope protein

LTR   LTR  

Ψ  

Ψ  

:  EncapsidaBon    signal  

LTR      Long  Terminal  Repeats    U3:  Promoter,  enhancer,  PolyA    5’R.  TranscripBon  start  signal  

Len4virus  

Gag (group antigens): matrix, core proteins Pol: reverse transcriptase: polymerase, RNaseH; integrase Env: envelope protein Tat: Transactivators Rev : nuclear export Vif, vpr, vpu, nef: replication and persistance of infection

RRE:  Rev  responsive  element.                    Binds  Rev  to  mediate                      nuclear  export    cPPT:  central  polypurine  track                        nuclear  import  

Retrovirus life cycle

Reverse  transcrip4on  of  the  RNA  genome  

PBS:  Primer  Binding  Site  PPT:  Polypurine  tract  

Nuclear  Import  

Integra4on  

The  preintegraBon  complex  (PIC)  :  Linear  viral  DNA,  integrase,  matrix  and  cellular  proteins    IntegraBon  is  not  random  and  favors  transcrip4onally  ac4ve  regions.  70%  in  genes.      Site  selecBon  is  governed  by  a  number  of  factors,  vector,  integrase,  chroma4n  structures,  etc.  The  PIC    complex  is  localized  to  genomic  DNA  through  an  interacBon  of  integrase  with  LEDGF/p75    in  HIV.  P75  thetering  integrase  and  chroma4n.    MuLV  preference  for  integra4on  in  transcrip4onal  start  sites,  CpG  islands  and  regulatory  regions  but  no  for  Len4vectors    Reduced  risk  of  inserBonal  mutagenesis  by    self-­‐inac4va4ng  SIN  vectors    

NPCNucleus

Active geneLEDGF

IN

LEDGF

LEDGF

HIV-1PIC

Cytoplasm

Enhancement of nuclear entry of PIC(?)

Stimulation ofintegration intochromatin (?)

Tethering of PIC on active gene (?)

Viral DNA structure and nuclear entry. The structure of viral DNA has been implicated in the nuclear import of HIV-1 PICs. The specific structural element involved is the central polypurine tract (cPPT, or central DNA flap), a short triple-stranded region of the viral DNA133,134. During synthesis of the viral DNA from the RNA genome, most of the RNA in the RNA–DNA hybrid is digested by the RNase-H activity of RT, whereas a short segment of polypurine tract (PPT) near the 3! terminus of the viral genome is resistant to digestion and is retained. Synthesis of (+)DNA (plus-strand) is then initiated from this PPT. In HIV-1, a second PPT is located centrally in the viral genome (cPPT). Once the 3! end of the (+)DNA reaches the 5! end of the cPPT, DNA synthesis proceeds by dis-placing the existing (+)DNA fragment and stops at a ter-mination sequence in the (–)DNA template. This results in a 99-bp triple-strand DNA structure in the centre of the viral DNA135,136. Mutation of the cPPT impairs HIV-1 replication135,137 and this sequence has been reported to be involved in nuclear accumulation of viral DNA in dividing and non-dividing cells133. Whereas insertion of the cPPT sequences into viral genomes clearly seems to improve the transduction efficiency of single-round lentiviral vec-tors64,138–140, the contribution of the DNA structure, par-ticularly in the nuclear import of replication competent HIV-1, remains controversial116,141,142. The slight effect of cPPT in single-round HIV-1 infection assays might be masked in spreading viral replication at high multi-plicity of infection; recent studies have shown that the absence of the cPPT significantly impairs HIV-1 replica-tion at low multiplicity of infection, which might more closely reflect HIV-1 replication in vivo143,144.

Cellular factors and nuclear entryThe ability of HIV-1 PICs to cross the intact nuclear envelope during interphase implicates the involvement of cellular active transport machineries.

Importin pathway and nuclear entry. Several compo-nents of the HIV-1 PIC, including the MA, Vpr and IN proteins, interact with members of the importin-" protein family100,102,108,111,145 (FIG. 3). Importin-" is a protein that forms a heterodimer with importin-#, and transports cargo molecules that contain an NLS across the nuclear envelope. After docking at an NPC through interaction of nucleoporin with importin-#, the importin cargo-protein complex is transported into the nucleus in an energy-dependent manner. In the nucleus, the cargo molecule is released into the nucleoplasm by the GTP-bound form of the GTPase Ran81 (BOX 1). Furthermore, Vpr protein interacts with the NPC102,146. A yeast two-hybrid study revealed that Vpr binds to the phenylalanine–glycine (FG) repeat of the NPC nucleoporin Pom121 (REF. 146). Interaction of Vpr with other nucleoporins has also been shown102,108,146. Nuclear translocation of Vpr does not require the Ran-mediated import pathway, suggesting that Vpr bypasses a common import system involved in receptor–cargo interaction and directly associates with NPCs106. The interaction of MA, Vpr and IN with importin or nucleoporin indicates that these viral proteins might

facilitate nuclear import. However, none of them is essential because chimeric viruses in which MA and IN are replaced with their murine leukaemia virus coun-terparts, or those lacking Vpr, can infect non-dividing cells105. A recent study using small interfering RNA (siRNA)-mediated gene silencing reported that deple-tion of the nucleoporin Nup98 impairs nuclear entry of HIV-1 DNA in growth-arrested cells147. This defect was also confirmed by reduced nuclear accumulation of viral DNA upon treatment with a specific inhibitor of Nup98 (REF. 147). Although an siRNA knockdown-based study implicated the involvement of importin 7 (REF. 148), a Ran-dependent nuclear-import receptor, in the active nuclear import of the HIV-1 PICs in both dividing HeLa cells and non-dividing macrophages, the role of impor-tin 7 as a mediator for nuclear entry of the PIC is unre-solved70. In addition to protein factors, cellular nucleic acids have been implicated in mediating nuclear import of HIV-1 PICs. Biochemical fractionation of cytoplasmic extracts that promote nuclear translocation identified a tRNA species as the active factor149.

LEDGF/p75 and nuclear entry. A recent study indicated that the HIV-1 IN lacks a transferable NLS and that the karyophilic property of IN is conferred by interaction with cellular factors35,123. LEDGF/p75, which interacts with HIV-1 IN, might be such a factor. LEDGF/p75 is a tran-scriptional regulator that associates with HIV-1 IN150 and

Figure 4 | LEDGF/p75 and nuclear entry of PICs. Several roles for lens-epithelium-derived growth factor (LEDGF/p75) have been proposed for human immunodeficiency virus 1 (HIV-1) DNA integration. LEDGF/p75 might regulate HIV-1 replication through the tethering of integrase protein (IN) and chromatin54. NPC, nuclear pore complex; PIC, pre-integration complex.

REVIEWS

192 | MARCH 2007 | VOLUME 5 www.nature.com/reviews/micro

Advantages                                                                  Drawbacks        

           Genome  size  8Kb                          IntegraBve  vectors,  Long  Term  expression                            Risk  InserBonal  mutagenesis    

Retroviral  vector      Len4viral  Vector  

Non  Human  origin                                                        Only  tranduces  dividing  cells                                                Low  Btres  

 Transduces  non-­‐dividing  cells                              Human  pathogen    High  Btres                              

Features  

Recombinant Retroviral Production

Vector              Viral  produc4on            Viral  4tra4on      Viral  transduc4on  of  target  cells    

Retroviral  Vector  construc4on  

 cDNA  HSVtk        HSVtk  P  

neo  BamH1  

LTR  

2  kb  

ψ LTR    

LTR  promoters  

SIN  vectors  Heterologous  promoters  

DNA  

DNA  

RNA  

RETROVIRAL Production •  Ecotropic: wide host range (MoMuLV) viruses from mice that only infect mice and closely related rodent. •  Amphotropic: infect rodents and other mammalian Packaging cell lines (3T3) Phoenix producer cell lines (293T)

•  Ph-Amphotropic

•  Ph-Ecotropic

•  PA317-amphotropic

•  AM-12-amphotropic

•  GP-E+86-ecotropic

•  Elevated titres. • Transient production

• Low titres. • Stable clones

Day 0

Seed 293T

Day 1

Transfection

Day 2

Change media

Day 3

SN1

Day4

SN2

RETROVIRAL  Produc4on  

Phoenix  293T  

PA317/tk/48  

PA317/tk/13  

10-­‐2   10-­‐3   10-­‐5  

Diluciones  del  sobrenedante  

       colonias  resistentes  

 dilución  del  sobrenedante  Título(virus/ml)  =  

 cDNA  HSVtk        HSVtk  P  

neo  BamH1  

LTR  

2  kb  

ψ LTR    

Retroviral  Titra4on  

]  ]  ]  ]  ]  

neomycin

ψ gen X neo

Target protein

 SN1  or  SN2  Fresh  or  from  frozen  stocks  +  Polybrene  or  protamine  sulfate  or  retronecBn          IncubaBon  or  spin  infecBon          One  or  two  cycles      

Target  Cell  Transduc4on  

5’  LTR+  Promotor,      Psi  ,  Cppt,      RRE,    SIN  3’LTR  Elements  required  in  cis:    

Len4viral  Vector  

VSV-G

Viral  concentra4on  (sucrose,  opBonal  )  70.000  x  g  -­‐  120.000  x  g    2h  

293  T  Cells  

Day 0

Seed 293T

Day 1

Transfection

Day 2

Change media

Day 3

SN1

Day4

SN2

Len4viral  Produc4on  

VSV-­‐G  

Physical  Titer.  PP    ELISA    an4-­‐HIVp24  capsid  protein  Immunoassay.  From  both  viral    Supernatants  and  purified  virus  

Infec4ous  Par4cles.  TU/ml    I.  Flow  cytometry.  EGFP  expressing  cells    II.  QPCR  .Standard  curve  of  DNA.                DNA  samples  from  transduced  cells  at  different  viral  diuBons.              Gag  oligos  .  Albumin  oligos  to  normalize  for  the  amount  of  genomic  DNA.  

Len4viral  Titra4on  

LTR miR155T LTR d2EGFP CMV

HeLa Control

1 MOI 10 MOI

Hipocampal cultures 5 days post-TD 0.5 MOI

Lv-EGFP

Len4viral  Transduc4on  

Kuroda et al. J. Gene Med. 2008

Lv-LacZ

In  vivo  Len4viral  Transduc4on.  Post-­‐mito4c  cells  

     Gene  Therapy  Advanced  Therapy    

1786  clinical  trials  

Gene  Therapy.  State  of  the  art  

Estudio de seguimiento de 9 años de media post-GT (entre 8 y 11) 9 pacientes: 4 Leucemia, 1 muerte. 7 pacientes incluyendo 3 supervivientes de leucemia tienen reconstituida sustancialmente la inmunidad. En 3 pacientes se les complementó con terapia con immunoglobulinas.

Bone marrow aspirates. CD34+ purification, culture with cytokines and transduced with RV under (GMP)  

All 10 patients are alive. Nine patients (but patient 8) are well with the duration of the follow-up ranging from 1.8 to 8 years. Patients 1 to 6 go to school regularly. 5 patients restored normal immune function. In the other five there was improvement in lymphocytes counts and functions. ADA gene transfer is superior to PEG- ADA

Bone marrow aspirates. CD34+ purification, culture with cytokines and transduced with RV. Infusion of CD34+ cells from bone marrow transduced with a Retroviral vector carrying the ADA gene.

Demyelinating disease of the CNS. . Mutations in a gene encoding for ALD protein, participates In the peroxisomal degradation of very long fatty acids in the oligodendrocytes and the microglia and disrupts Myelin mantainance. ( replacement of microglia cells from donor bone marrow

Ex-vivo mediated lentiviral ALD transfer in CD34+ cells

Follow-up of 33 months, 21 months no transfusion

ProSavin®  

ProSavin  uBlises  Len4Vector®  system  to  deliver  the  genes  AADC  (aromaBc  amino  acid  decarboxylase),  TH  (tyrosine  hydroxylase)    and  CH1  (GTP-­‐cyclohydrolase  1).      These  genes  reprogramme  transduced  cells  to  manufacture    and  secrete  dopamine.  

Oxford  BioMedica  (LSE:  OXB)    Phase  I/II  trial  of  ProSavin,  its  novel  gene  therapy  for  the  treatment  of  Parkinson’s  disease.  PaBents  treated  at  the  low  iniBal    dose  have  completed  two  years.  Improvement  in  motor  funcBon  (UPDRS  III  ‘OFF’  score).      The  maximum  improvement  was  30%  and  the  average  was  20%  rela4ve  to  pa4ents’    pre-­‐treatmentmotor  func4on.     June 2010. Higher dose group (one year follow-up), maximum 56% improvement. May 2011. Higher dose (six month follow-up), motor improvement, average 43% maximum 61% one patient.

Reprogramación Celular-Terapia Génica

Fibroblasto de paciente alteración genética

Lentivirus con el gen correcto

Célula Madre Embrionaria Alteración (IPS)

Célula Madre Embrionaria Corregida

Diferenciación Celular

PNAS,  2006  

Qasim,  Mol  Ther.  2010  

Next  Genera4on  Retroviral  Gene  Therapy  

Anabel  José  Victoria  Maliandi  Xevi  Bofill  Eneko  Villanueva  Ana  Mato  Maria  Rovira  Ana  Boullosa  Luciano  Sobrevals  Xavier    Altafaj  Núria  Andreu  Laura  García  Jon  OrBz  Daniel  Abate-­‐Daga  Meritxell  Huch  Marta  Riera  Anna  Cascante  Meritxell  Carrió  

Ramon  Alemany  ICO,  Barcelona    Olga  Millan,Raúl  Herrance  IAT,  Barcelona    Adela  Mazo  Universitat  de  Barcelona  

Collaborators  

Anabel  

Eneko  

Ana  

Cris4na  Maria  

Xevi  

Vicky  

Ana