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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